Physiol Rev
83: 377– 415, 2003; 10.1152/physrev.00025.2002.
From Genes to Integrative Physiology: Ion Channel
and Transporter Biology in Caenorhabditis elegans
KEVIN STRANGE
Departments of Anesthesiology, Molecular Physiology and Biophysics, and Pharmacology,
Vanderbilt University Medical Center, Nashville, Tennessee
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I. Introduction: From Genes to Integrative Physiology
II. The Nematode Caenorhabditis elegans
A. Establishment of C. elegans as a model organism
B. C. elegans biology
C. Tools and experimental strategies for studying ion channel and transporter biology in C. elegans
III. Integrative Physiology of Caenorhabditis elegans Membrane Transport Processes
A. Worms have feelings too: DEG/ENaC cation channels
B. Of mice and worms: new insights into ClC anion channel physiology
C. Staying regular: the IP3 receptor
D. Cleaning house: ABC transporters
E. Life and death and things in between: TRP and related channels
F. Getting the message out: neurotransmitter transporters and ligand-gated channels
G. Complex simplicity: K⫹ channels
H. Conclusions and future perspective
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Strange, Kevin. From Genes to Integrative Physiology: Ion Channel and Transporter Biology in Caenorhabditis elegans.
Physiol Rev 83: 377– 415, 2003; 10.1152/physrev.00025.2002.—The stunning progress in molecular biology that has
occurred over the last 50 years drove a powerful reductionist approach to the study of physiology. That same progress
now forms the foundation for the next revolution in physiological research. This revolution will be focused on integrative
physiology, which seeks to understand multicomponent processes and the underlying pathways of information flow from
an organism’s “parts” to increasingly complex levels of organization. Genetically tractable and genomically defined
nonmammalian model organisms such as the nematode Caenorhabditis elegans provide powerful experimental advantages for elucidating gene function and the molecular workings of complex systems. This review has two main goals. The
first goal is to describe the experimental utility of C. elegans for investigating basic physiological problems. A detailed
overview of C. elegans biology and the experimental tools, resources, and strategies available for its study is provided. The
second goal of this review is to describe how forward and reverse genetic approaches and direct behavioral and
physiological measurements in C. elegans have generated novel insights into the integrative physiology of ion channels
and transporters. Where appropriate, I describe how insights from C. elegans have provided new understanding of the
physiology of membrane transport processes in mammals.
I. INTRODUCTION: FROM GENES TO
INTEGRATIVE PHYSIOLOGY
Physiology is defined as the function of a living organism
and its parts. The enormous breadth of physiological research,
which ranges from the study of single molecules to whole
organisms, reflects the inherent complexity of life. The stunning progress in molecular biology that occurred during the last
50 years drove a powerful reductionist approach in physiological research. That same progress, however, now forms the
foundation for the next revolution in the field. This revolution
www.prv.org
will be focused on integrative physiology, which seeks to understand multicomponent processes and the underlying pathways of information flow from an organism’s “parts” up
through increasingly complex levels of organization. Identification of the genes and proteins involved in specific physiological
processes will lead ultimately to the integrated molecular understanding of underlying signaling and metabolic pathways,
macromolecular protein assemblies, and functional interactions of organelles, cells, tissues, and organs.
This review has two main goals. The first goal is to
describe the experimental utility of nonmammalian model
0031-9333/03 $15.00 Copyright © 2003 the American Physiological Society
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KEVIN STRANGE
II. THE NEMATODE CAENORHABDITIS
ELEGANS
A. Establishment of C. elegans
as a Model Organism
The establishment of C. elegans as a model system
for fundamental biological research began in 1963 with
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the efforts of Sydney Brenner, a molecular biologist then
at the Medical Research Council (MRC) Laboratory of
Molecular Biology in Cambridge, United Kingdom. In a
letter to Max Perutz, the head of the laboratory, Brenner
(37) stated that “it is widely realized that nearly all the
’classical’ problems of molecular biology have either been
solved or will be solved in the next decade” and that the
“future of molecular biology lies in the extension of research to other fields of biology, notably development and
the nervous system.”
The extraordinary successes that had been achieved
at that time in defining the molecular bases of biological
processes in bacteria suggested to Brenner that a similar
approach would work in more complex organisms. He
told Perutz that he wanted to “define the unitary steps of
development using the techniques of genetic analysis.” To
tackle this problem, Brenner felt that he would need to
identify a metazoan animal that he could “microbiologize”
and handle in much the same way as bacteria and viruses.
Brenner concluded his letter by stating that he wanted to
“tame a small metazoan organism to study development
directly.”
Despite criticism from other molecular biologists
that his approach was too “biological,” Brenner was asked
to submit a formal proposal to the MRC. He did so in
October 1963, proposing to study the genetics of differentiation in the nematode Caenorhabditis briggsae.1 The
possible utility of nematodes in genetic research had first
been noted by Dougherty and Calhoun (71). In Brenner’s
opinion, Caenorhabditis provided numerous experimental advantages for identifying genes involved in development. The animal has a short life cycle, produces large
numbers of offspring by sexual reproduction, and can be
cultured easily in the laboratory. Sexual reproduction
occurs by self-fertilization in hermaphrodite worms or by
mating with males, which makes Caenorhabditis exceptionally useful for genetic studies. Self-fertilization allows
homozygous worms to breed true and greatly facilitates
the isolation and maintenance of mutant strains. It is also
a handy feature if mutant animals are paralyzed or uncoordinated since reproduction does not require movement
to find and mate with a male. Mating with males, however,
is essential for moving mutations between strains. Finally,
Caenorhabditis is a highly differentiated animal but is
comprised of ⬍1,000 somatic cells and therefore provides
a tractable system for studies of metazoan cellular function, development, and differentiation.
1
C. elegans eventually replaced C. briggsae as the species of
choice. This was due in part to the ability to obtain high-quality electron
micrographs from C. elegans, which eventually led to the development
of a complete wiring diagram for the nervous system (348). In addition,
there appears to have been early confusion over the taxonomic classification of the two species and of the identification of the species
present in laboratory stocks (273).
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organisms for investigating basic physiological problems.
What is a model organism? The physiologist and Nobel
laureate August Krogh believed that there is an ideal species
or “model system” in which almost every biological problem
can be studied most readily, a belief that is often referred to
as the “Krogh Principle” (175, 176). Krogh would define a
model organism as any organism that provides experimental
characteristics that are optimal for the study of a physiological process.
In the postgenome sequencing era, the term model
organism has taken on a more explicit meaning. Model
organisms are “simpler,” genomically defined organisms
that are easy to grow in the laboratory, produce large
numbers of offspring, and have relatively short life cycles
making them suitable for detailed genetic analyses. These
characteristics allow more rapid, efficient, and economical manipulation, and hence understanding, of gene function than what is possible in “complex” organisms.
Despite the narrowing of the definition of model organism, the Krogh Principle applies, perhaps more than ever, to
the field of integrative physiology. If one wishes to define the
genes and genetic pathways that give rise to complex physiological processes, doing so in a simple model system that
is genetically tractable, and where it is relatively easy to
manipulate gene expression makes sound experimental
sense. Organisms such as Saccharomyces, Drosophila, and
even the plant Arabidopsis are powerful experimental systems for addressing physiological problems common to all
eukaryotes. The nematode Caenorhabditis elegans is an
exquisite example of the experimental advantages provided
by genetically tractable model organisms. This review provides a detailed overview of C. elegans biology and the
experimental tools, resources, and strategies available for its
study.
Ion channels and transporters play fundamental roles
in organelle, cell, organ, and whole organism function.
These roles range from regulation of organelle pH, cell
volume, and membrane excitability to control of systemic
salt and water balance and behavior. The second goal of
this review is to describe how forward and reverse genetic approaches and direct behavioral and physiological
measurements in C. elegans have generated novel insights
into the integrative physiology of ion channels and transporters. I will also describe how insights from C. elegans
have provided new understanding of the physiology of
membrane transport processes in mammals.
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
B. C. elegans Biology
1. Natural history and life cycle
C. elegans is a member of the phylum Nematoda.
Nematodes, or roundworms, are some of the most numer-
ous and widespread of all animals and are found in virtually all habitats. The phylum contains free-living species
as well as species that parasitize plants and other animals.
In addition to its extraordinary utility as a model for basic
biological research, detailed understanding of all aspects
of C. elegans function has major economic and medical
implications. Approximately one-half the world’s human
population is infected with parasitic nematodes (205), and
parasitic plant nematodes cause an estimated $80 billion
in crop damage annually (287).
Nematodes range in size from ⬍1 mm to over 35 cm
in length. C. elegans is a free-living soil nematode ⬃1 mm
long. The life strategy of C. elegans is well adapted for
survival in soil environments where food and water availability, temperature, populations of predators, and many
other variables can change constantly and dramatically. It
is a voracious feeder that, as noted by Riddle et al. (273),
consumes “anything that fits in its mouth.” To outgrow
competitors, C. elegans produces large numbers of offspring and rapidly depletes local food resources.
Adult C. elegans are predominantly hermaphroditic
with males making up ⬃0.1% of wild-type populations.
Self-fertilized hermaphrodites produce ⬃300 offspring,
whereas male-fertilized hermaphrodites can produce over
1,000 progeny.
Under optimal laboratory conditions, the average life
span of C. elegans is 2–3 wk. The life cycle is rapid (Fig. 1).
At 25 °C, embryogenesis, the period from fertilization to
hatching, occurs in ⬃14 h. Postembryonic development occurs in four larval stages (L1–L4) that last a total of ⬃35 h.
FIG. 1. Life cycle of Caenorhabditis elegans
grown at 25°C on agar plates seeded with Escherichia coli. Eggs are laid ⬃5 h after fertilization, and hatching occurs ⬃9 h later. [From
Jorgensen and Mango (153), with permission
from Nature Reviews Genetics, copyright 2002
Macmillan Magazines Ltd.]
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Brenner concluded his proposal with the following
statement, “To start with we propose to identify every cell
in the worm and trace lineages. We shall also investigate
the constancy of development and study its control by
looking for mutants.” The statement is audacious and will
surely amuse anyone who has struggled through the National Institutes of Health funding system. Nevertheless, it
was Brenner’s audacity and the good judgement of the
MRC that established C. elegans as a model organism.
C. elegans researchers have accomplished Brenner’s
goal of tracing the lineage of every nematode cell and
identifying genes responsible for development. In 2002,
Sydney Brenner shared the Nobel Prize in Physiology or
Medicine with John Sulston and H. Robert Horvitz for
their discovery of genes in C. elegans that regulate organ
development and programmed cell death. However, as
discussed below, the extraordinary experimental power
of C. elegans has been exploited well beyond studies of
developmental biology. This tiny animal has provided and
undoubtedly will continue to provide important insights
into fundamental biological problems such as ageing,
RNA-mediated gene silencing, cell cycle control, sensory
physiology, and synaptic transmission.
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When food supply is limited, dauer larvae form after
the second larval molt. Dauer larvae do not feed and have
structural, metabolic, and behavioral adaptations that increase life span up to 10 times and aid in the dispersal of
the animal to new habitats. Once food becomes available,
dauer larvae feed and continue development to the adult
stage (243, 297).
2. Laboratory culture
3. Anatomy
Like all nematodes, C. elegans has an unsegmented,
cylindrical body that tapers at both ends. The body wall
consists of tough collagenous cuticle underlain by hypo-
FIG. 2. Schematic diagrams showing anatomical features of an adult C. elegans hermaphrodite (top) and male
(bottom). [From Sulston and Horvitz (307), with permission from Academic Press.]
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The standard C. elegans laboratory strain is Bristol
N2. Other strains are also used and offer certain experimental advantages. For example, the Bergerac strain exhibits a high rate of spontaneous mutation due to the
activity and high copy number of the Tc1 transposon (see
sect. IIC3D) (220). The Hawaiian strain CB4856 possesses
a uniformly high density of single nucleotide polymorphisms that greatly facilitate genetic mapping (349).
Culture of C. elegans in the laboratory is simple and
relatively inexpensive (187). Animals are typically grown
in petri dishes on agar seeded with a lawn of E. coli as a
food source. C. elegans can also be grown in mass quantities using liquid culture strategies and fermentor-like
devices. Worm stocks are stored frozen in liquid nitrogen
indefinitely with good viability. The ability to store C.
elegans frozen dramatically simplifies culture strategies
and reduces costs associated with handling and maintaining wild-type and mutant worm strains.
dermis, muscles, and nerves. A fluid-filled body cavity or
pseudocoel separates the body wall from internal organs.
Body shape is maintained by hydrostatic pressure in the
pseudocoel (Fig. 2).
Newly hatched L1 larvae have 558 cells. Additional
divisions of somatic blast cells occur during the four
larval stages eventually giving rise to 959 somatic cells in
mature adult hermaphrodites and 1,031 in adult males.
The lineage of somatic cells in C. elegans is largely invariant. This invariance combined with the ability to visualize
by differential interference contrast (DIC) microscopy
cell division and development in living embryos, larvae
and adult animals has made it possible to describe the fate
map or cell lineage of the worm (307, 308).
Despite the small cell number, C. elegans exhibits an
extensive degree of differentiation. Many physiological functions found in mammals have nematode analogs. This high
degree of complexity and small total cell number provides a
remarkably tractable experimental system for studies of
differentiation, cell biology, and cell physiology.
A) SKIN. The worm “skin” or hypodermis is an epithelium that underlies the cuticle. Hypodermal cells secrete
the cuticle, provide a substrate for cell and axon migration, and function as a storage site for lipids and other
molecules (126, 347). The hypodermis is also involved in
phagocytosis of apoptotic cells (275, 308) and may have
an osmoregulatory function (248). Certain types of hypodermal cells function as blast cells and give rise postembryonically to new cell types such as neurons (308). Most
hypodermal cells are multinucleate.
B) MUSCLE. C. elegans is a particularly powerful model
for defining muscle physiology, structure, molecular biology, and development (reviewed in Refs. 219, 343). The
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
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The excretory cell is a large, H-shaped cell that sends
out processes both anteriorly and posteriorly from the
cell body (e.g., see Fig. 5). A fluid-filled excretory canal is
surrounded by the cell cytoplasm. The basal pole of the
cell faces the pseudocoel while the apical membrane
faces the excretory canal lumen. Gap junctions connect
the excretory cell to the hypodermis, suggesting an interaction between the two cell types important for whole
animal osmoregulation.
An excretory duct connects the excretory canal to
the outside surface of the worm. The duct is formed from
cuticle that is continuous with the animal’s exoskeleton.
A duct cell surrounds the upper two-thirds of the duct and
a pore cell surrounds the lower one-third.
The excretory cell is a single-cell “epithelium” that
appears to secrete salt, water, and waste products into the
excretory canal. Duct cells may also play an important
role in solute and water transport. The apical surface area
of the duct cell is greatly amplified by extensive invaginations, and the cytoplasm is filled with mitochondria.
Nelson et al. (230) have suggested that the duct cell may
be involved in selective solute reabsorption. If this is the
case, the nematode excretory and duct cells are analogous to the acini and ducts of mammalian secretory epithelia such as the salivary gland, sweat glands, and pancreas. The excretory cell may be a particularly valuable
model for defining molecular mechanisms of stimulussecretion coupling (reviewed in Refs. 130, 162) and ion
channel and transporter trafficking and polarization.
E) DIGESTIVE TRACT. The digestive tract of C. elegans consists of a pharynx, intestine, and rectum. C. elegans is a filter
feeder, and the pharynx is a muscular organ that pumps
food into the pharyngeal lumen, grinds it up, and then moves
it into the intestine. The pharynx is formed from muscle
cells, neurons, epithelial cells, and gland cells (3).
A pharyngeal-intestinal valve connects the pharynx to
the intestine. Twenty epithelial cells with extensive apical
microvilli form the main body of the intestine (186). Intestinal epithelial cells secrete digestive enzymes and absorb
nutrients. The intestine functions as one of the major storage
organs in the body. Intestinal cells are filled with numerous
granules that likely contain lipids, proteins, and carbohydrates (173). The intestine also produces yolk proteins and
secretes them into the pseudocoel where they are then
taken up by oocytes (111, 116, 165).
The rectum is made up of five epithelial cells and is
connected to the intestine via the intestinal-rectal valve. A
sphincter muscle wraps around the valve and controls its
opening. The sphincter muscle, an anal depressor muscle,
and two muscle cells that wrap around the posterior
intestine control defecation.
F) GONAD. The gonad of adult hermaphrodites consists
of two identical U-shaped arms connected via spermatheca to a common uterus (116, 131, 140) (e.g., see Fig.
8A). The gonad arms are surrounded by thin epithelial
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worm has a number of readily observable characteristics
that allow rapid screening for mutations in genes involved
in muscle function. Even mutations that cause severe
locomotion defects can be studied readily and in detail
because hermaphrodites self-fertilize and locomotion is
unnecessary for reproduction.
C. elegans possesses both striated and nonstriated muscles. Striated body wall muscles are the most numerous
muscle cell type. They are arranged in longitudinal bands
along the body wall and are responsible for locomotion.
Nonstriated muscles are associated with the pharynx, intestine, anus, and the hermaphrodite uterus, gonad sheath, and
vulva. These muscles are responsible for pharyngeal pumping, defecation, ovulation and fertilization, and egg laying.
Specialized nonstriated muscles are located in the tail of
male worms and function in mating.
C) NERVOUS SYSTEM. The nervous system of adult hermaphrodites contains 302 neurons and 56 glial and support cells. Males have 381 neurons and 92 glial and support cells. White et al. (348) have reconstructed and
mapped the connectivity of the entire hermaphrodite nervous system using serial electron microscopy. Most of the
differences between the male and hermaphrodite nervous
system are found in the male tail, which plays an important role in mating. Partial reconstruction of the tail nervous system has been performed (306).
The nervous system of C. elegans is generally thought
of as being divided into the pharyngeal and central nervous systems. Twenty neurons innervate and regulate the
activity of the pharynx. These neurons are connected to
the central nervous system by two interneurons.
Neuronal processes of the central nervous system are
oriented as bundles that run along the hypodermis in
longitudinal cords or circumferential commissures. The
ventral and dorsal nerve cords emanate from the circumpharyngeal nerve ring. Interneurons and motor neurons
make up the ventral nerve cord. The dorsal nerve cord is
formed largely of ventral cord motor neuron axons that
enter via commissures. Sense organs that respond to
chemicals, temperature, mechanical force, and osmolality
are located primarily in the head and tail.
An important feature of the C. elegans nervous system is that only three neurons, CANL, CANR and M4, are
required for viability under laboratory conditions. The
CAN (excretory canal) neurons run along the excretory
canals (see sect. IIB3D) and may play an important role in
regulating systemic salt and water balance. M4 is a pharyngeal motor neuron that controls isthmus peristalsis
and thus controls feeding (12). The nonessential nature of
most neurons for viability provides an enormous advantage for mutagenesis studies of nervous system function.
D) KIDNEY. The worm “kidney” consists of three cell
types: the excretory cell, the duct cell, and the pore cell
(230). Destruction of any of these cells by laser ablation
causes the animal to swell with fluid and die (231).
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C. Tools and Experimental Strategies for Studying
Ion Channel and Transporter Biology
in C. elegans
1. Cell physiology
A) TARGETED KILLING OF IDENTIFIED CELL TYPES. A powerful
way to assess the physiological role of a specific nematode cell type is to destroy the cell and characterize the
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effect on developmental events and whole animal phenotype. Laser ablation or microsurgery has been used extensively to identify cell function and cell-cell developmental interactions in C. elegans (18). Identified cell types
are destroyed using a nitrogen-laser-pumped dye laser.
The laser beam is focused onto the cell of interest through
a high numerical aperture objective lens on a compound
microscope equipped with DIC optics.
It is also possible to genetically target cells for killing.
Maricq et al. (204) first used this approach to kill neurons
expressing the glutamate receptor gene glr-1. The glr-1
promoter was fused to the mec-4(d) open reading frame
and DNA transformation carried out using standard methods (see sect. IIC3B). mec-4(d) encodes a mutant DEG/
ENaC cation channel that is thought to be constitutively
active (72). Constitutive channel activation presumably
results in excessive cation influx into the cell, which leads
to cell swelling and necrotic cell death (115). Driscoll and
co-workers (121) have demonstrated that ectopic expression of mec-4(d) induces degeneration of a wide variety
of cell types. Targeted cell killing has also been carried
out using human caspase-1 to induce apoptotic cell death
(362) and diptheria toxin (87).
B) ELECTROPHYSIOLOGY OF DIFFERENTIATED SOMATIC CELLS. In
vivo measurement of physiological processes such as ion
channel and transporter activity in C. elegans is technically demanding and time consuming. Most somatic cells
are quite small, and a tough pressurized cuticle surrounding the animal limits access to the cells for direct functional measurements.
To patch-clamp neurons, Lockery and co-workers
(109, 198) developed the so-called “slit worm preparation”
(Fig. 3A). Animals are immobilized by gluing them with
cyanoacrylate glue onto a glass coverslip coated with a
thin layer of agarose. Exposure of the neurons involves
cutting a small slit in the worm with a glass dissecting
needle. Groups of neurons containing several cell bodies
emerge through the cut. Different types of neurons can be
identified using cell-specific green fluorescent protein
(GFP) reporters (see sect. IIC3C).
Richmond and co-workers (271, 272) developed a
“filleted worm” preparation to study body wall muscle
electrophysiology (Fig. 3B). As with the slit worm preparation, animals are immobilized on agarose pads with
cyanoacrylate glue. A lateral incision in the cuticle is then
made to expose the body wall muscles.
A filleted preparation was described recently by
Brockie et al. (38) that allows access to and partially
preserves the spatial organization of neurons in the head.
Briefly, worms are glued to Sylgard-coated coverslips and
the cuticle along the length of the pharynx is slit with a
fine glass needle. A flap of cuticle is then retracted and
pinned to the Sylgard (Fig. 3C).
The C. elegans pharynx has proven to be an important model system for identifying the genetic basis of
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cells termed sheath cells. The distal portion of each arm
contains germline nuclei that give rise to sperm and oocytes. Each nucleus is surrounded by cytoplasm and an
incompletely formed plasma membrane. The nuclei in
turn are arranged in a monolayer that surrounds a central
core of cytoplasm (e.g., see Fig. 8A).
The germline nuclei population is maintained by mitosis occurring in the distal-most tip of each gonad arm.
As germ cells progress down the arm, they exit the mitotic
cell cycle and undergo meiosis. Formation of an intact
germ cell plasma membrane, a process termed cellularization, occurs in the proximal arm of the gonad. During
the fourth larval stage, germ cells differentiate into ⬃150
spermatids/gonad arm. These spermatids develop into
mature sperm that are stored in the spermatheca.
In adult worms, germ cells differentiate into oocytes.
Oocytes are arranged in the proximal gonad arm in a
single-file fashion. Once formed, oocytes undergo oogenesis, which is a period of intense biosynthetic activity and
rapid and massive oocyte growth.
Oocytes remain in diakinesis of prophase I until they
reach the most proximal position in the gonad arm. During the late stage of oogenesis, an oocyte located immediately adjacent to the spermatheca undergoes meiotic
maturation (e.g., see Fig. 8, A and B). Within 5– 6 min after
maturation is initiated, the oocyte is ovulated into the
spermatheca where it is fertilized. Completion of the meiotic divisions occurs in the uterus and is followed by
embryogenesis. In a mature hermaphrodite, ovulation occurs once every 20 – 40 min. Unmated hermaphrodites
produce ⬃300 progeny over a 3-day period when grown
under standard laboratory conditions (116, 131, 341).
The testis, seminal vesicle, and vas deferens form the
male gonad (131, 168). The testis is U-shaped and is
formed from two distal tip cells that surround the germ
cells. As in the hermaphrodite gonad, germ cell nuclei at
the distal end of the testis undergo mitosis. Germ nuclei
farther away enter prophase of meiosis I. Spermatogenesis begins in the proximal end of the testis.
The seminal vesicle is formed by 20 secretory cells.
Spermatocytes enter the vesicle and undergo two meiotic
divisions to form mature spermatozoa. Spermatozoa are
stored in the seminal vesicle. The vas deferens is comprised of 30 cells and functions as a valve that controls
release of sperm during ejaculation.
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
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excitable cell function. Raizen and Avery (260) developed
a simple extracellular recording method for monitoring
electrical transients induced by changes in the membrane
potential of pharyngeal muscles. Briefly, the head of a
worm is sucked into a fluid-filled glass pipette and electrical connections between the pipette fluid and bath are
made with silver/silver chloride electrodes (Fig. 3D). During an action potential, the basal membrane of the pharPhysiol Rev • VOL
ynx depolarizes. Excitation of the pharyngeal muscles
causes current to flow across the apical membrane of the
pharynx, out of the worm’s mouth, and back into the
pseudocoelom through the cuticle and hypodermis. These
electrical transients are termed the electropharyngeogram (EPG). The EPG equivalent circuit and a typical
EPG from a wild-type worm are shown in Figure 3D.
Analysis of EPG in mutant animals has been particularly
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FIG. 3. Preparations used for electrophysiological measurements in C. elegans. A: schematic diagram and differential
interference contrast (DIC) micrograph of “slit worm” preparation used for patch clamping of C. elegans neurons.
Neuron expressing a green fluorescent protein (GFP) reporter is shown in green. (DIC image kindly provided by Dr.
Shawn Lockery.) B: schematic diagram and fluorescence micrograph of “filleted worm” preparation used for patch
clamping of C. elegans body wall muscles. GFP reporters are expressed in ventral nerve cord and body wall muscles.
Scale bar is 50 ␮m. [From Richmond and Jorgensen (272), with permission from Nature Publishing Group.] C: schematic
diagram and fluorescence micrograph of “filleted head” preparation used for patch clamping neurons. AVA interneurons
are identified using GFP expressed under the control of the nmr-1 promoter. [From Brockie et al. (38), with permission
from Elsevier Science.] D, top: schematic diagram and equivalent circuit of preparation for recording electropharyngeogram (EPG). [From Avery et al. (13), with permission from Academic Press.] Bottom: EPG from a wild-type worm.
The first upward spike (E) indicates muscle depolarization that initiates contraction. Large downward spike (R) is
muscle repolarization that precedes relaxation. Inhibitory postsynaptic potentials (IPSPs) generated by the M3 motor
neuron occur when the muscle is depolarized. [From Dent et al. (69), with permission from Oxford University Press.]
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D) CELL CULTURE. Because of technical hurdles, largescale cell culture methods have not been widely exploited
until recently for the study of C. elegans. Culturing differentiated cells from larvae and adult C. elegans is probably
not technically feasible because of difficulties in removing
the animal’s cuticle and in dissociating cells. In contrast,
embryonic cells can be isolated relatively easily (see sect.
IIC1C). Early attempts at large-scale culture of C. elegans
embryonic cells were described by Bloom (32). This pioneering work demonstrated that morphological differentiation occurred in these cells. However, Bloom noted
significant problems with cell survival, attachment of cells
to the growth substrate, cell differentiation, and reproducibility of the methods. Initial attempts to patch-clamp
cultured cells were unsuccessful. Buechner et al. (41)
have also reported that cultured C. elegans embryonic
cells undergo morphological differentiation resembling
that of neurons and muscle cells.
Recently, Christensen et al. (53) described methods
that allow the robust, large-scale culture of C. elegans
embryonic cells. Isolated embryonic cells differentiate
within 24 h into the various cell types that form the newly
hatched L1 larva. Expression of a number of cell-specific
GFP reporters and molecular markers is similar to that
observed in the intact animal (Fig. 4). Cultures can be
generated from animals of almost any genetic background, and the cells can be readily patch clamped (53,
54) and loaded with ion-sensitive fluorescent dyes (A.
Estevez and K. Strange, unpublished observations). Addition of double-stranded RNA (dsRNA) to the culture medium induces dramatic knockdown of targeted gene expression in cultured cells. Fluorescence-activated and
magnetic-activated cell sorting can be used to enrich cultures for certain cell types. Enriched cultures can in turn
be used for cell-specific biochemical, molecular, DNA
microarray, and proteomic studies. C. elegans cell culture
should be particularly valuable for physiologists interested in the molecular bases of ion channel and transporter function. When combined with other C. elegans
tools, primary cell culture provides new opportunities for
developing an integrated systems level understanding of
eukaryotic ion channel and transporter biology.
E) QUANTITATIVE MICROSCOPY. C. elegans is well-suited
for quantitative, in vivo microscopy studies of ion channel
and transporter activity. The embryo eggshell and cuticle
of larvae and adults are transparent, making it possible to
observe and quantitate cell biological events and physiological processes using bright-field and fluorescence microscopy. Differential interference contrast microscopy
produces superb images of embryos, larvae, and adult
animals. Larvae and adult worms are easily immobilized
for imaging studies by anesthetizing with buffer containing 0.1% tricaine and 0.01% tetramisole or by gluing them
with cyanoacrylate glue onto a glass coverslip or slide
coated with a thin layer of agarose.
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useful for defining the molecular bases of cell excitability
(e.g., see sect. IIIG1).
The pharynx can also be isolated easily by cutting the
animal transversely behind the pharyngeal bulb. After
cutting, body wall muscles contract, retracting the cuticle
covering the head and exposing the pharynx. The isolated
pharynx can be impaled with glass microelectrodes (64,
65, 93, 245).
C) ELECTROPHYSIOLOGY OF GERM CELLS AND EARLY EMBRYOS.
The first reported patch-clamp studies on C. elegans were
carried out on spermatocytes and spermatids (200). Like
those of other nematodes, C. elegans spermatozoa are
nonflagellated ameboid cells (187a). Sperm cells can be
isolated easily by cutting male worms with a needle close
to the vas deferens.
Nematode oocytes are also relatively easy to isolate.
Briefly, individual nematodes are cut behind the pharyngeal bulb using a 26-gauge needle. The extruded gonad is
then detached by making another cut in front of the
spermatheca. Isolated gonads contract and eject oocytes
through the cut end. It is also possible to microdissect
sheath cells away from the oocytes using glass micropipettes (279). Oocytes are readily patch clamped (54, 279)
and can be loaded with voltage-, Ca2⫹-, and pH-sensitive
fluorescent dyes (C. Boehmer, E. Rutledge, and K.
Strange, unpublished observations).
Mass quantities of oocytes can be isolated from the
fer (fertilization defective)-1 worm strain (8, 303). The
fer-1 hermaphrodites produce normal sperm and are selffertilizing at 18°C. However, at 25°C, the sperm do not
develop properly and are incapable of fertilizing oocytes.
Unfertilized oocytes are ovulated and accumulate in the
uterus where they continue to undergo DNA replication
and become polyploid. Induction of oocyte laying is triggered by treating worms with serotonin and levamisole
(8) or by gentle sonication (303). Released oocytes are
separated from adult worms and debris by filtering
through mesh screens. In our experience, mass-isolated
oocytes are difficult to patch clamp in the conventional
whole cell mode (E. Rutledge and K. Strange, unpublished
observations). However, they may be useful for other
types of patch-clamp measurements as well as radioisotope flux studies.
Embryonic cells can be isolated easily and in large
quantities. Adult nematodes are treated with an alkaline
hypochlorite solution, which lyses the cuticle (187). Eggs
released by this treatment are pelleted by centrifugation
and washed in saline. Eggshells are removed by resuspending pelleted eggs in a chitinase solution (79). After
digestion of the eggshell, early embryo cells are dissociated by passing the embryo suspension through a 23gauge needle. As with oocytes, embryo cells can be patch
clamped (54) and loaded with ion-sensitive fluorescent
dyes (M. Christensen and K. Strange, unpublished observations).
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
385
Proteins encoded by transgenes offer the potential to
monitor membrane voltage and intracellular and intraorganelle ion levels in intact worms. For example, Ca2⫹sensitive cameleons (216, 217) have been used to measure
intracellular Ca2⫹ transients in pharyngeal muscle (160).
GFP has been used as an optical sensor of membrane
voltage changes induced by mechanical stimulation of C.
elegans touch neurons in vivo (161). Other transgeneencoded cameleon proteins and GFPs may be useful for in
vivo measurements of Cl⫺ (178), Ca2⫹ (16, 226), pH (170,
Physiol Rev • VOL
197), and signaling molecules that control ion transport
processes such as cAMP (359), cGMP (134), and Ca2⫹calmodulin (247). It is also possible to microinject oocytes (286) and intestinal cells (63) in vivo with ionsensitive fluorescent dyes.
Fluorescence resonance energy transfer (FRET)
(159) using the GFP color mutants CFP (cyan) and YFP
(yellow) (214, 217) may be a powerful way to test for and
further characterize protein-protein interactions in vivo in
C. elegans that have been predicted by genetic screens
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FIG. 4. Micrographs of cultured C.
elegans embryonic cells expressing cellspecific GFP reporters. A: myo-3::GFP
expression in cultured body muscle
cells. myo-3 encodes a myosin heavy
chain isoform expressed in body muscles. The myo-3 reporter strain expresses two GFPs with peptide signals
that target them to either the nucleus
(arrows) or mitochondria (arrowheads).
B: cultured mechanosensory neuron expressing mec-4::GFP. mec-4 encodes a
degenerin-type ion channel subunit expressed largely in neurons that respond
to gentle body touch. C: cultured cholinergic
motor neurons expressing unc-4::GFP.
unc-4 encodes a transcription factor. D:
cultured neuron expressing opt-3::GFP.
OPT-3 is a H⫹/oligopeptide transporter
expressed in glutamatergic neurons. E:
combined DIC and fluorescence micrograph of an unc-119::GFP-expressing
neuron. unc-119 encodes a novel protein
that is expressed in all neurons. F: combined DIC and fluorescence micrograph
of an unc-4::GFP-expressing cholinergic
motor neuron physically interacting with
a body wall muscle cell. All scale bars
are 10 ␮m. [From Christensen et al. (53),
with permission from Elsevier Science.]
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and yeast two-hybrid and biochemical assays. This approach has been used recently to demonstrate a direct
interaction between voltage-activated Ca2⫹ channels and
calmodulin in transfected mammalian cells (85).
2. Genetics and the genome
Physiol Rev • VOL
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A) FORWARD GENETIC SCREENING. The development of C.
elegans as an experimental system was driven largely by
the relative ease of performing forward genetic screens
for identification of the complement of genes responsible
for observable phenotypes. The utility and power of genetic screening depends on the ability to assay a phenotype of interest. For a detailed and illuminating discussion
of screening assays in C. elegans, the reader is referred to
a recent review article by Jorgensen and Mango (153).
Once a screening assay is developed, animals are
mutagenized, typically by the alkylating agent ethyl methane-sulfonate (EMS). Mutant animals are then isolated
and the mutated gene identified by mapping, rescue, and
cloning strategies (153). In addition to identifiying genes
involved in a specific biological process, forward genetic
screens can also provide important information on protein structure/function.
Mutant animals can be further mutagenized to produce double mutants. The phenotype of a double mutant
could be suppressed, enhanced, or similar to the phenotype of animals with a single mutation. Mutations that
enhance or suppress a phenotype may reside in genes
distinct from the one mutated in the original screen.
These extragenic mutations imply that the enhancer and
suppressor genes interact with the first mutated gene.
Genetic interactions indicate that gene products function
in a common pathway. They also suggest the possibility of
direct interactions between gene products (i.e., proteinprotein interactions) (153). Ion channels, transporters,
and associated regulatory machinery have been identified
in genetic screens of various processes such as locomotion (4, 179, 206, 210, 261, 264), gonad development (345),
mechanosensory (203, 314, 315) and osmotic avoidance
behavior (58), chemotaxis and thermotaxis (225), resistance to fluoride ions (313), defecation (63, 64), egg laying
(81, 152, 181), toxin and heavy metal sensitivity (39, 70,
92), programmed cell death (352), neuronal degeneration
(322), and abnormal catecholamine levels (75).
B) THE GENOME. The C. elegans genome is diploid and
consists of 97 ⫻ 106 base pairs of DNA organized onto 5
autosomal chromosomes and one X sex chromosome.
Recombinational mapping of mutant genes was first reported by Brenner (36) in 1974. In 1984, assembly of a
clone-based physical map of the genome was begun (61).
Work from numerous laboratories has subsequently led to
the development of a detailed genome map with tight
correlation between genetic and physical mapping data
(60). The physical map of the worm genome is comprised
of overlapping cosmid and yeast artificial chromosome
(YAC) clones. These clones are freely available to
the research community from the Sanger Center in
Cambridge, United Kingdom (http://www.sanger.ac.uk/
Projects/C_elegans/). The availability of these clones from
a centralized resource has greatly facilitated genetic research in C. elegans. For example, identification of mutated genes is carried out by mapping the mutation to a
genetic interval and then rescuing the mutant animal by
microinjection of candidate cosmids (see sect. IIC3B).
The development and testing of technology required
for the sequencing and understanding of the human genome was critically dependent on the analysis of genomes
of less complex organisms such as C. elegans. Funding for
sequencing of the worm genome was obtained in 1990
from the National Institutes of Health and the MRC in the
United Kingdom. The sequencing project was a collaborative effort between the Sanger Institute in the United
Kingdom and the Genome Sequencing Center at Washington University School of Medicine in St. Louis, Missouri.
Sequencing of the worm genome was largely completed
by the end of 1998 (318) and was the first sequence
obtained for a multicellular organism. The genome contains ⬃20,000 predicted protein- and RNA-encoding genes
(132). As of this writing, ⬍10% of these genes have been
studied experimentally.
To aid in gene identification, two major projects are
ongoing. The C. elegans expressed sequence tag (EST)
project undertaken by Kohara and co-workers (http://
www.ddbj.nig.ac.jp/c-elegans/html/CE_INDEX.html) has
tag sequenced over 60,000 cDNAs. These ESTs have identified more than 9,000 worm genes.
Vidal and co-workers (266) (http://worfdb.dfci.harvard.edu/) have undertaken an effort to clone all C. elegans open reading frames (ORFs), which they term the
“ORFeome.” ORFs are amplified from a cDNA library
using specific primers and then sequenced to generate
ORF sequence tags (OSTs). These OSTs support the existence of at least 17,300 C. elegans genes (266). To expedite functional analysis of the ORFs, they are cloned
using the Gateway recombination cloning strategy (124,
336). This approach has the advantage of allowing directional cloning of PCR products into a reference vector and
subsequent subcloning into various vector backgrounds
without the use of restriction enzymes and ligases. As
such, recombination cloning is amenable to automation
and high-throughput analysis of gene function.
Gene identification in C. elegans has also been facilitated by development of the Intronerator (158) (http://
www.cse.ucsc.edu/⬃kent/intronerator/), which is a series
of internet tools for analyzing gene structure. Intronerator
provides alignments of cDNAs with genomic sequence
and a catalog of alternatively spliced genes.
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
3. Functional genomics
Physiol Rev • VOL
B) CREATION OF TRANSGENIC ANIMALS. DNA transformation
in C. elegans is relatively straightforward and was first
reported by Stinchcomb et al. (300). Fire (88) and Mello et
al. (213) have described methods for producing and maintaining transgenic worm lines. Briefly, transforming DNA
is microinjected into the distal end of the hermaphrodite
gonad. Heritable DNA transformation occurs by extrachromosomal transformation, nonhomologous integration, or homologous integration. Spontaneous homologous integration is extremely rare. Formation of multicopy extrachromosomal arrays is the most frequent way
in which transforming DNA is inherited. Transformation
by extrachromosomal arrays is often transient. Integration of transgenes and generation of stable transgenic
lines is commonly carried out by gamma irradiation of
transformed worms (212).
Recently, Praitis et al. (255) described the use of
microparticle bombardment to create integrated transgenic lines in C. elegans. This approach is technically less
demanding than microinjection and reportedly produces
single- and low-copy chromosomal DNA insertions resulting in more reliable transgene expression. Low-copy integrated lines also exhibit expression of transgenes in cells
of the germline. Multicopy extrachromosomal arrays are
often not expressed in germ cells due to the activity of
gene silencing processes (e.g., Ref. 157).
C) IDENTIFYING THE CELLULAR LOCALIZATION OF GENE PRODUCTS. Determining where and when a gene is expressed
can provide important clues to its function. Gene expression patterns in C. elegans have been determined by in
situ hybridization (30, 292, 311) and by creation of transgenic animals expressing lacZ (89) or GFP (49) reporter
genes. Translational GFP reporters have been particularly
useful for defining the cellular location and trafficking of
proteins in C. elegans (e.g., Refs. 38, 78, 172). Examples of
GFP reporter localization of ion channel and transporter
proteins are shown in Figure 5. Databases of gene expression patterns in C. elegans are accessible online (http://
nematode.lab.nig.ac.jp/db/index.html, http://129.11.204.
86:591/default.htm, http://www.wormbase.org/).
D) GENE KNOCKOUT. Targeted gene inactivation by homologous recombination has not been feasible in C. elegans (252). Instead, the relative ease of culturing C.
elegans in large numbers and the ability to store worms
frozen has led to the development of so-called “targetselected gene inactivation methods.” This approach involves inducing random deletion mutations in a population of worms. Mutagenized worms are subdivided into
small pools, typically in 96-well culture plates. These
pools are screened by PCR for a deletion mutation in a
target gene. Once a pool containing the desired mutation
is detected, it is subdivided and PCR screening is repeated
until single worms carrying the deletion are isolated.
Zwaal et al. (365) were the first to use target-selected
gene inactivation methods. Deletion mutations were
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A wealth of powerful molecular tools and online
databases are available to study ion channel and transporter functional genomics in C. elegans. Conceptually,
functional genomic studies can be divided into two approaches. The gene-driven approach focuses on identification, cloning, and characterization of genes. Gene characterization involves expression and structure-function
studies and determination of where and when a gene is
expressed in the organism. The phenotype-driven approach seeks to determine the physiological roles of
genes from the cellular to whole animal level and to
identify the mechanisms that regulate protein function.
A) IDENTIFICATION OF TRANSMEMBRANE PROTEIN FAMILIES IN
THE C. ELEGANS GENOME. An important starting point in functional genomics research is the identification and classification of genes from raw genome sequence. Identification of genes encoding membrane transport proteins provides the basis for discovery of new transporters and
channels, for understanding channel and transporter evolution, for elucidating structure-function relationships,
and for determining the physiological roles and regulation
of transport processes.
Membrane transport physiologists are well acquainted with the use of hydrophobicity analysis for predicting transmembrane helices and, in theory, such an
approach could be used for predicting membrane proteinencoding genes. The reliability of hydropathy methods,
however, is limited (150, 169). Hidden Markov models
(HMMs) with high accuracy for predicting transmembrane protein topology, have been developed recently
(177, 325). HMMs are probabilistic models applicable to
linear problems such as genome sequence analysis. Krogh
et al. (177) (http://www.cbs.dtu.dk/services/TMHMM/)
used an HMM to identify membrane-spanning proteins in
the genomes of several organisms including C. elegans.
Approximately 30% of the genes in the worm genome
encode proteins with one or more predicted transmembrane helices.
Ian Paulsen and his group at the Institute for
Genomic Research (TIGR) have analyzed the complement
of predicted and known transport protein-encoding genes
in the genomes of C. elegans and several other organisms
(http://www.biology.ucsd.edu/⬃ipaulsen/transport/).
These genes are classified into families according to the
Transport Commission (TC) system (http://tcdb.ucsd.edu/
tcdb/). The TC system is a scheme for classifying transport proteins based on functional and phylogenetic information and is analogous to the Enzyme Commission system for enzyme classification (281, 282). Paulsen’s
analysis has revealed that more than 750 genes or ⬃4% of
the worm genome encode proteins thought to be involved
in ATP-dependent, secondary active, and passive, channel-mediated transport processes.
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screened for in cultures of mut (mutator)-2 worms in
which endogenous transposons are actively “jumping”
and inserting randomly into the genome (358). Bessereau
et al. (28) have recently demonstrated that the Drosophila
transposon Mos1 can be expressed and can function as a
mutagen in the C. elegans genome. The use of heterologous transposons offers a number of advantages for forward genetic studies (see Ref. 28).
Jansen et al. (147) demonstrated that random deletion mutations in C. elegans could be generated in a
large-scale manner using chemical mutagens. Highthroughput methods for isolation of C. elegans deletion
mutants have been described in detail (195). A C. elegans
Knockout Consortium (http://elegans.bcgsc.bc.ca/knockout.shtml) consisting of three collaborating laboratories
is currently working to produce strains possessing deletion mutations in all identified worm genes. Deletion
strains are freely available to all investigators, and requests for knockout of a specific gene can be made online
to the consortium (http://elegans.bcgsc.bc.ca/cgi-bin/submit
_ko.pl).
E) RNA-MEDIATED GENE INTERFERENCE. One of the truly
extraordinary experimental advantages of C. elegans is
the relative ease by which gene function can be disrupted
using dsRNA-mediated gene interference (RNAi). RNAi
was discovered originally in C. elegans (90), but it now
seems likely that the phenomena of posttranscriptional
transgene silencing and quelling described earlier in
Physiol Rev • VOL
plants and fungi, respectively, are occurring via dsRNAregulated mechanisms (33, 253, 342). RNAi is thought to
be a component of endogenous gene silencing mechanisms that protect cells against viral infection and transposon activity (22, 33, 142, 253, 293, 342). It may also play
a regulatory role in developmental events (112, 171).
The precise molecular mechanisms that mediate RNAi
are unknown. However, a number of important studies performed over the last 2 years have begun to provide a working model. dsRNA readily crosses cell membranes by unknown mechanisms. Once inside the cell, dsRNA is cleaved
rapidly into small fragments ⬃21–25 bp in length. These
small interfering RNAs (siRNAs) are the active agents and
are thought to associate with a complex of proteins that
includes RNases. The antisense strand on each 21–25mer
base pairs with homologous single-stranded mRNA. After
base pairing has occurred, the mRNA is destroyed by RNase
activity (242, 355, 360). Recent studies have suggested that
siRNAs may also serve as primers to transform target mRNA
into dsRNA via the action of an RNA-dependent RNA polymerase (191, 234, 296). These newly synthesized dsRNAs are
then cleaved into additional siRNAs. It has been estimated
that just a few molecules of dsRNA in a cell are sufficient to
dramatically knockdown targeted gene expression (90).
dsRNA disrupts targeted gene expression in a variety
of invertebrate animals, plants, protozoans, and fungi (22,
33, 142, 253, 293). However, exposure of differentiated
mammalian cells to long dsRNAs frequently triggers an
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FIG. 5. Examples of transgenic nematodes expressing
cell-specific GFP reporters. A: fluorescence (left) and DIC
(right) micrographs of a nematode expressing GFP in six
dopaminergic neurons. GFP expression is driven by the
promoter for the dopamine transporter (DAT). CEP and
ADE dopaminergic neurons are indicated. Fluorescence
micrograph is a 3-dimensional reconstruction generated
by confocal microscopy. [From Nass et al. (227), copyright
2002 National Academy of Sciences USA.] B: fluorescence
(left) and DIC (right) micrographs of a nematode expressing GFP in the H-shaped excretory cell (EC). GFP expression is driven by the clh-4b promoter. CLH-4b is a ClC
chloride channel homolog. [From Nehrke et al. (229).]
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
Physiol Rev • VOL
Genetics. In addition, investigators can send RNA samples to the Kim laboratory (http://cmgm.stanford.edu/
⬃kimlab/wmdirectorybig.html) where they will be hybridized with the microarray and the data deposited online
in the Stanford Microarray Database (http://genomewww5.stanford.edu/MicroArray/SMD/).
C. elegans microarrays and DNA chips have been
used to examine developmental changes in gene expression (129, 151) and to identify oocyte- and sperm-enriched
and sex-related genes (151, 269). Kim et al. (164) recently
assembled data from multiple microarray experiments
and developed an expression map of coregulated worm
genes. Microarray technology in combination with cell
culture and sorting methods (53) now make it feasible to
identify cell-specific gene expression patterns under a
variety of experimental conditions. For example, Chalfie
and co-workers (361) have identified touch-cell specific
genes by hybridizing a C. elegans microarray with amplified mRNA from FACS-enriched GFP-expressing touch
neurons.
G) THE INTERACTOME: DEVELOPMENT OF A GENOME-WIDE PROTEINPROTEIN INTERACTION MAP. Protein-protein interactions play critical roles in most biological processes. Understanding these
interactions is thus an essential component of functional
genomics research. As in all organisms, protein-protein interactions can be identified in C. elegans by coimmunoprecipitation, pull-down assays, and yeast two-hybrid screens.
Most physiologists are familiar with these experimental
strategies and would typically use them when fishing for
partners that interact with a single protein of interest. However, with the availability of complete genome sequences,
large-scale, high-throughput yeast two-hybrid screens have
been proposed as a way to map genome-wide protein-protein interactions (96, 183, 330). Genome-wide protein interaction maps can lead to new understanding of gene function
in several ways. First, biological insights can be gained into
proteins with unknown functions by linking them to proteins that operate in characterized biological pathways. Genome-wide screens can also identify novel interactions occurring between two or more proteins that function in a
common physiological process, and they may identify new
interactions and hence functions for previously characterized proteins.
Identification of genome-wide protein-protein interactions would clearly provide an extraordinary wealth of
data from which experimentally testable hypotheses concerning the function and regulation of proteins, not only
in C. elegans, but also in other organisms, could be proposed. Walhout et al. (335) (http://vidal.dfci.harvard.edu/
main_pages/interactome.htm) recently assessed the feasibility of generating a protein-protein interaction map for
the C. elegans genome. As a starting point, these investigators focused on 27 genes known to be involved in worm
vulval development. PCR fragments were inserted into
yeast two-hybrid vectors using the Gateway recombina-
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interferon response leading to widespread, nonselective
inhibition of gene expression and induction of apoptosis
(156, 350). Recently, several groups have reported that the
interferon response can be circumvented and specific
gene silencing induced by transfecting mammalian cells
with siRNAs (e.g., Refs. 43, 80, 122, 133, 236, 237, 244, 305,
354). It should be noted, however, that gene silencing by
RNAi in mammalian cells is experimentally more complicated than in invertebrate cells. Mammalian cells must be
transfected with siRNAs, and their silencing effect is relatively short-lived. In addition, the effectiveness of RNAi
in silencing a mammalian gene may be dependent on the
region of the mRNA that is targeted by the siRNAs (e.g.,
Ref. 133; see Ref. 211 for a review on the use of siRNA in
mammalian cells).
The ability of dsRNA to readily cross C. elegans cell
membranes is particularly advantageous for experimental
disruption of gene expression. dsRNA can be microinjected anywhere in a worm and its effect will spread
throughout the animal’s body (90). In addition, RNAi can
be induced by feeding worms bacteria making dsRNA
(155, 320, 321) or by soaking them in dsRNA solutions
(310). The systemic spread of gene silencing by dsRNA is
dependent in part on a putative transmembrane protein
SID-1 (351). Worms can also be engineered with inducible
transgenes that are transcribed into dsRNA (317). In C.
elegans cell culture, RNAi can be triggered simply by
adding dsRNA to the culture medium (53; see also papers
describing RNAi in cultured Drosophila cells, Refs. 42, 56,
262, 326).
RNAi is being used with great success to carry out
large-scale, high-throughput reverse genetic screening of
the C. elegans genome. Four separate groups have performed RNAi on ⬃33% of the animal’s genes using microinjection (106, 250), feeding (94), and soaking (201) strategies. Of the ⬃6,275 genes examined to date, ⬃888 give
rise to observable whole animal phenotypes when their
expression is disrupted by dsRNA. As noted by Kim (163),
the phenotypic analysis that has been carried out is at a
“shallow level.” Nevertheless, these important studies
have assigned some level of function to a significant portion of the C. elegans genome and have provided the
foundation for the generation and testing of countless
new hypotheses.
F) DNA MICROARRAYS. DNA microarrays and DNA chips
allow gene expression patterns to be monitored on a
genome-wide scale. Affymetrix has produced DNA chips
that collectively contain ⬃98% of the worm genome. Kim
and co-workers (151, 269) have produced a microarray
that contains ⬃94% of the worm’s genes. The microarray
was generated by PCR amplification of genomic DNA.
Primer sequences are available online (http://cmgm.stanford.edu/⬃kimlab/primers.12–22–99.html), and the complete set of primers can be purchased from Research
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Physiol Rev • VOL
I) VALUE OF GENOME-SCALE DATA COLLECTION.
It is not uncommon to hear or read criticisms that are leveled at genomewide investigative strategies such as microarray analyses,
database mining, large-scale RNAi screens, and development of protein-protein interaction maps. For example, a
recent editorial published in the Journal of General Physiology (7) stated that the “sheer amount of information”
becoming available in the postgenomic era has forced a
“shift toward high throughput screens, where the traditional
notions of hypothesis-driven experiment and definitive
mechanistic insight become elusive.” Hypotheses are
formed by asking questions about observations. One can
make observations, for example, by measuring the kinetics
of solute flux across a membrane or the electrophysiological
properties of an ion channel, or by performing high-throughput, genome-scale screens. These data collection methods
differ only in scale. The torrent of data made available by
genome sequencing and genome level functional screens is
unprecedented in biological research. Although these data
may not be useful to all scientists, they are invaluable to
anyone interested in integrative biology.
Rather than driving a shift away from hypothesisdriven investigation, genome-wide data and high-throughput screens now make it possible to develop and test
hypotheses in ways unimaginable just a few years ago.
Figure 6 illustrates this point schematically. Classical,
hypothesis-driven investigations typically focus on one or
a few proteins. Data on the function and regulation of
these proteins are collected and hypotheses proposed to
guide further experimentation and data collection. When
these data are integrated with relevant genome-scale data,
the results are inevitably the development of new hypotheses and deeper insights into biological function. For
example, imagine that one is studying the role of binding
protein X that regulates ion channel Y. A genome-scale
protein interaction map might identify protein X as part of
a cluster of interacting proteins (for example, see Refs.
35, 66). It would be reasonable to postulate that these
interacting proteins are components of a signaling pathway and/or scaffolding complex that regulates channel Y.
Similarly, microarray analyses might reveal that expression of binding protein X is coregulated with several other
signaling/scaffolding proteins under specific physiological
conditions, in different tissues, and/or at different times
during development. Coregulation of these proteins
would again suggest that they are components of a regulatory complex. Tissue-, physiological-, and developmental-dependent changes in binding protein X expression
might suggest new functional roles for it as well as for
channel Y. Physiologists interested in the molecular workings of complex systems from macromolecular protein
assemblies to whole organisms should revel in data collected from genome-wide analyses.
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tion cloning strategy (124, 336) described in section IIB2.
Both the PCR and cloning steps are amenable to automation and thus can be scaled up to perform genome-wide
interaction screens. Their screen of vulval development
genes detected two novel interactions and ⬃50% of previously described interactions. When the vulval development genes were screened against a C. elegans cDNA
library, 124 potential interactors were identified. Fifteen
of these interactors had been identified previously in genetic screens, and 109 were new interactors predicted
from genome sequence.
More recently, Davy et al. (66) began developing a
protein-protein interaction map for the 26S proteasome
from C. elegans. As a starting point, they searched the C.
elegans genome for orthologs of human and yeast 26S
proteasome subunits and related proteins. Thirty-two potential orthologs were identified, and 30 of these were
PCR-amplified. Each of these ORFs was then screened
against a C. elegans cDNA library using the high-throughput yeast two-hybrid assay described by Walhout et al.
(336). Ninety-four potential interactors were identified.
These included interactions reported for other organisms,
novel interactions between proteasome subunits, and
novel proteasome interacting proteins.
Boulton et al. (35) have combined large-scale proteinprotein interaction mapping and RNAi feeding experiments to identify genes involved in the DNA damage
response (DDR). BLAST searches were used initially to
identify 75 putative C. elegans DDR orthologs. Sixty-seven
of these orthologs were screened by high-throughput
yeast two-hybrid assay, and 165 potential interactors were
detected. The DDR orthologs and the interacting proteins
were then subjected to RNAi feeding experiments (see
sect. IIC3E). These studies confirmed that 12 of the 75
predicted orthologs were involved in the DDR response,
and they identified 11 novel DDR genes. Taken together,
the high-throughput screens carried out by Walhout et al.
(335), Davy et al. (66), and Boulton et al. (35) have identified dozens of new genes and protein-protein interactions that provide the foundation for the development and
testing of at least an equal number of new hypotheses.
H) ONLINE ACCESS TO C. ELEGANS BIOLOGY. The “worm
community” is well known for its open sharing of data and
reagents. As discussed above and summarized in Table 1,
an extraordinary wealth of information on C. elegans is
available publicly online. Indeed, the worm community
was an early pioneer in the use of the internet for electronic data sharing.
WormBase (http://www.wormbase.org/) is a particularly noteworthy database. It provides an exhaustive catalog
of worm biology. The database contains the worm genome,
the complete cell lineages of hermaphrodite and male
worms, genetic maps, gene expression patterns, RNAi phenotypes, worm genetics, and an atlas of worm anatomy.
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ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
TABLE
1. Publicly available online C. elegans databases and resources
Site
Information and Services Available
URL
Anatomy
The Center for C. elegans
Anatomy
Aid and train C. elegans investigators in electron
microscopy; curators of an online atlas of worm
anatomy
Genomics
http://www.aecom.yu.edu/wormem/
Sanger Institute
Sequenced the C. elegans genome in collaboration
with the Genome Sequencing Center at the
Washington University School of Medicine, St.
Louis, MO; provide cosmid and YAC clones to
investigators
Repository of information on genetics, genomics,
and biology of C. elegans including gene
sequence, gene expression patterns, RNAi
phenotypes, genetic maps and worm anatomy
Tools for analyzing gene structure and RNA
splicing
Database of C. elegans open reading frames
Database of C. elegans expressed sequence tags
http://www.sanger.ac.uk/Projects/C elegans/
(Contact Dr. Alan Coulson for clones)
WormBase
ORFeome project
C. elegans EST Database
http://www.cse.ucsc.edu/⬃kent/intronerator/
http://worfdb.dfci.harvard.edu/
http://www.ddbj.nig.ac.jp/c-elegans/html/CE IND
Transport proteins
Transport Commission Database
Genomic Comparisons of
Membrane Transport Systems
Center for Biological Sequence
Analysis
Comprehensive classification system for
membrane transport proteins
Databases of known and predicted transporters
and channels in the genomes of C. elegans and
other organisms
Online hidden Markov model for predicting
transmembrane helices
http://tcdb.ucsd.edu/tcdb/
http://www.biology.ucsd.edu/⬃ipaulsen/transport/
http://www.cbs.dtu.dk/services/TMHMM/
Gene knockout
The C. elegans Gene Knockout
Consortium
Produce null alleles of all known C. elegans
genes; provide knockout animals to
investigators
Fraser et al. (94)
Gönczy et al. (106)
Piano et al. (250)
Maeda et al. (201)
RNAi analysis of chromosome I
RNAi analysis of chromosome III
RNAi analysis of genes expressed in worm ovary
Large-scale analysis of gene function induced by
soaking worms in dsRNA
Vidal lab INTERACTome project
Genome-scale protein-protein interaction mapping
http://vidal.dfci.harvard.edu/main pages/interacto
Kim lab C. elegans microarrays
Microarray
Performs DNA microarray experiments;
microarray databases
http://cmgm.stanford.edu/⬃kimlab/wmdirectorybig.
http://elegans.bcgsc.bc.ca/knockout.shtml
Large-scale RNAi screens
http://www.wormbase.org/
http://worm-srv1.mpi-cbg.de/dbScreen/
http://www.rnai.org/
http://nematode.lab.nig.ac.jp/db/rnai s/index.html
Protein-protein interactions
Gene expression patterns
The Nematode Expression
Pattern Database
The Hope Laboratory
Expression Pattern Database
WormBase
In situ hybridization database
http://nematode.lab.nig.ac.jp/db/index.html
lacZ and GFP reporter expression database
http://129.11.204.86:591/default.htm
Exhaustive database includes lacZ and GFP
reporter expression patterns
http://www.wormbase.org/
Caenorhabditis Genetics Center
Collect, maintain, and distribute worm strains;
maintain a C. elegans bibliography; publish and
distribute the Worm Breeder’s Gazette
Caenorhabditis elegans WWW
Server
A Caenorhabditis elegans
Survival Kit
Links to many useful sites including an online
catalog of methods
Links to many useful sites
Worm strains
http://biosci.umn.edu/CGC/CGChomepage.htm
Miscellaneous
http://elegans.swmed.edu/
http://members.tripod.com/C.elegans/c elegans Int
GFP, green fluorescent protein.
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Intronerator
http://www.wormbase.org/
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III. INTEGRATIVE PHYSIOLOGY OF
CAENORHABDITIS ELEGANS MEMBRANE
TRANSPORT PROCESSES
In this section, I review the functional biology of membrane transport processes in C. elegans. It is important to
stress that my discussion of the topic is not meant to be
comprehensive. Instead, my intention here is to illustrate the
experimental power of genomically defined and genetically
tractable nonmammalian model organisms such as C. elegans for the study of integrative membrane transport physiology. I do this by describing how the molecular workings
of complex physiological processes in which ion channels
and transporters play key roles have been and are continuing to be elucidated in C. elegans through a combination of
forward and reverse genetic screening methods and direct
physiological, behavioral, and cell biological measurements.
Where appropriate, I discuss how studies on nematodes
have provided new insights into ion channel and transporter
physiology in mammals.
A. Worms Have Feelings Too: DEG/ENaC Cation
Channels
1. Mechanosensation in C. elegans
All organisms have the ability to detect and respond
to mechanical force. Mechanical signaling plays an important role in a wide variety of biological processes ranging
from osmoregulation to hearing and locomotion (105,
118). Remarkably, relatively little is known about the
molecular basis of mechanosensation.
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C. elegans has proven to be an invaluable model
system in which to identify genes encoding proteins required for mechanosensory processes. Mechanical signals
likely regulate a variety of nematode behaviors including
locomotion, foraging, and the ability of the animal to
detect and respond to touch. The best understood mechanosensory behavior is the avoidance response initiated
when the animal is touched lightly on the body with an
eyelash hair (45). Gentle touch to either the posterior or
anterior body causes the animal to move away in a forward or backward motion.
Laser ablation studies identified five neurons required for the response to gentle body touch (46). These
“touch” neurons are also called microtubule cells because
their processes contain a bundle of microtubules, each of
which is formed from 15 protofilaments (47, 48). Touch
neurons include the anterior ventral microtubule cell
(AVM) and the anterior and posterior lateral microtubule
cells left, right (ALML/R and PLML/R, respectively). The
posterior ventral microtubule cell (PVM) is also considered to be a touch neuron because its morphology is the
same as that of the AVM, ALML/R, and PLML/R and
because it expresses touch neuron specific genes. However, ablation of this neuron does not appear to alter
touch sensitivity (46).
2. Identification of genes responsible for touch
neuron function
Genes required for touch neuron mechanosensory
functions were identified by mutagenizing worms and
assessing their ability to respond to gentle body touch (44,
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FIG. 6. Integration of data from hypothesis-driven investigations and genome-wide
analyses. The “classical” approach is envisioned as being primarily hypothesis driven
and focused on the function and regulation
of one or a few proteins involved in a biological process. Genome-wide analyses focus primarily on large-scale, high-throughput collection and collation of data. When
relevant data from the two approaches are
integrated, the result is inevitably deeper
insight into biological processes and the
development of new hypotheses.
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
Physiol Rev • VOL
type protease inhibitor domains, and a glutamic acid-rich
region, suggesting that it may play a role in mediating
protein-protein interactions.
3. Functional interaction of MEC-2,
MEC-6, MEC-4, and MEC-10
Goodman et al. (108) demonstrated recently that
MEC-4 and MEC-10 form functional Na⫹ channels and
that MEC-2 plays an important role in regulating their
activity. When expressed alone or together in Xenopus
oocytes, wild-type MEC-4 and MEC-10 do not give rise to
detectable Na⫹ currents. However, amiloride-sensitive
currents are detected when channel proteins containing
the “degenerin” mutation, MEC-4d and MEC-10d, are coexpressed. Coexpression of MEC-2 with MEC-4d and
MEC-10d increases current ⬃40-fold. Currents are also
detected, albeit at a much lower level, when MEC-2 is
expressed with wild-type MEC-4 and MEC-10.
Genetic studies suggest that MEC-2 interacts with the
MEC-4/MEC-10 channel complex (113, 138). In support of
this finding, Goodman et al. (108) demonstrated that antiMEC-2 antibodies immunoprecipitated MEC-4d and
MEC10d expressed in oocytes. Taken together, these and
other studies indicate that MEC-4 and MEC-10 form a heteromeric ion channel that is regulated by MEC-2 (Fig. 7).
The central domain of MEC-2 (amino acids 114 –363)
is 64% identical to human stomatin. Of 54 mutant alleles of
mec-2 detected in genetic screens, more than half map to
this region, indicating that it is particularly important for
mechanosensory neuron function (139). Consistent with
this idea, the central domain alone is capable of stimulating amiloride-sensitive currents, albeit significantly less
than the full-length protein. Human stomatin induces a
similar increase in amiloride-sensitive currents. Taken
together, these studies provide the first direct evidence
that stomatins regulate ion channel activity. MEC-2 has no
effect on membrane trafficking of MEC-4d or MEC-10d,
suggesting that it functions to increase channel open
probability, open time, and/or unitary conductance (108).
Degenerin function in vivo is dependent on MEC-6
(50, 102, 138, 193, 294, 316), a 377-amino acid protein with
homology to vertebrate paraoxonases (PONs) (51a).
PONs are involved in phospholipid metabolism and interact with cell plasma membranes and high-density lipoprotein (200a). MEC-6 is expressed in numerous cell types
including touch neurons (51a). Sequence and membrane
topology analyses suggest that MEC-6 has a single transmembrane-spanning domain, a short cytoplasmic NH2 terminus, and a large extracellular COOH terminus (51a).
When coexpressed with MEC-4d/MEC-10d or MEC-4d/
MEC-10d and MEC-2, MEC-6 increases amiloride-sensitive Na⫹ currents ⬃30- and ⬃200-fold, respectively (51a).
MEC-6 has no effect on membrane trafficking of MEC-4d
or MEC-10d (51a), suggesting that the protein, like MEC-2,
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45). Touch-insensitive mutant worms are mechanosensory abnormal, and the genes responsible for this abnormality are termed mec. Approximately 15 mec genes have
been identified to date. Several of these genes are required for normal touch neuron development. At least
eight mec genes encode proteins that have been postulated to form a mechanosensitive ion channel complex
(203, 314, 315).
The mec-4 and mec-10 genes encode ion channel forming proteins that share significant homology with epithelial
Na⫹ channels or ENaCs (5, 203). The first ENaC-like channel
encoding gene identified in C. elegans was deg-1 (degeneration of certain neurons). Gain-of-function mutations in
deg-1 cause various nematode neurons to swell and degenerate (50); hence, the protein encoded by the gene was
termed a “degenerin.” One particular mutation in deg-1 results in the substitution of alanine at position 707 to valine
(102). This amino acid is located at a putative extracellular
site adjacent to the second transmembrane domain. Mutation of the equivalent alanine in mec-4 and mec-10 also
induces neuronal degeneration (72, 138). Heterologous expression studies of Drosophila and vertebrate DEG/ENaC
homologs have demonstrated that this mutation induces
constitutive channel activation (1, 2, 51, 334).
Twenty-five members of the DEG/ENaC family have
been identified in the C. elegans genome (315). The channels are expressed in a wide variety of cell types including
neurons, muscle cells, intestine, and hypodermis (313,
314). Three of these channels, UNC-105, UNC-8, and
DEL-1, have been suggested to play roles in processes
thought to be regulated by mechanical force (101, 193,
241, 294, 316).
The mec-7 (117, 288) and mec-12 (97) genes encode
␤- and ␣-tubulins, respectively. Mutations in these genes
disrupt the formation of the 15 protofilament microtubules in touch neurons (48, 97, 289). The mec-2 gene
encodes a stomatin-like protein (139). Mammalian stomatin is a 31-kDa integral membrane protein that associates
with the cytoskeleton (298) and lipid raft proteins (285).
Red blood cells in patients with hereditary stomatocytosis
are deficient in stomatin and exhibit abnormalities in
shape and monovalent cation permeability (68). Interestingly, stomatin is coexpressed with ENaC-type channels
in mammalian mechanosensory neurons (95, 103, 202).
Touch neuron processes are surrounded by an extracellular matrix or “mantle,” which attaches the cells to the
cuticle (45). Mantle proteins play important roles in touch
neuron mechanosensation. The mec-1 gene encodes a
protein required for formation of the mantle (45), and
mec-5 encodes a specialized collagen that is secreted by
hypodermal cells (74). Genetic analyses suggest that
MEC-5 may interact with MEC-4 and MEC-10 (113). The
mec-9 gene encodes a protein secreted by touch neurons
that interacts genetically with MEC-5 (74). MEC-9 contains six epidermal growth factor-like repeats, five Kunitz-
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FIG. 7. Model of a mechanosensory ion channel
complex in C. elegans touch neurons. Mechanical distortion of the cuticle is postulated to gate the MEC-4/
MEC-10 channel complex open by displacing it with
respect to cytoskeletal and extracellular matrix attachment points.
4. Is there a vertebrate equivalent of the putative touch
neuron mechanosensory ion channel complex?
The proposed involvement of C. elegans DEG/ENaC
channels in touch sensation has stimulated the search for
Physiol Rev • VOL
similar functional roles of this channel class in vertebrates. ENaCs reconstituted in lipid bilayers (14, 143) or
expressed in fibroblasts (167) have been reported to be
activated by membrane stretch. However, mechanical gating of ENaCs is not observed under all conditions (15).
Recently, Ma et al. (199) reported that extracellular ATP
acting through a phospholipase C-dependent purinergic
pathway masks ENaC mechanosensitivity.
ENaCs have recently been suggested to function as
components of the arterial baroreceptor mechanotransducer. Drummond et al. (73) demonstrated that ␥-ENaC is
expressed in baroreceptor nerve terminals that innervate
the carotid sinus and aortic arch. In addition, they demonstrated that baroreceptor nerve activity and baroreflex
control of blood pressure were blocked by benzamil, a
potent inhibitor of ENaC activity.
Welsh and co-workers (259) recently made an intriguing observation that directly linked DEG/ENaC channels to vertebrate touch perception. DEG/ENaC homologs
have been identified in the vertebrate brain and are referred to as brain Na⫹ channels or BNaCs (100, 259).
Knockout of BNaC1 in mice reduces the sensitivity of
low-threshold, rapidly adapting hairy skin mechanoreceptors, which are stimulated by hair movement. Immunostaining for BNaC1 demonstrated that the channel was
localized to nerve endings surrounding hair follicles (257).
These findings suggest that BNaC1 is a component of a
vertebrate mechanoreceptor analogous to the DEG channels in nematode touch neurons.
Another BNaC-type channel, DRASIC (dorsal root
acid sensing ion channel; Ref. 333), may also function as
part of a mechanoreceptor complex. DRASIC is expressed in mouse dorsal root ganglion sensory neurons
that respond to mechanical stimuli such as light touch,
muscle stretch, and noxious touch (258). Knockout of the
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increases macroscopic current by increasing channel
open probability, open time, and/or unitary conductance.
Heterologous expression and coimmunoprecipitation assays have demonstrated that MEC-6 interacts physically with MEC-2, MEC-4, and MEC-10 (51a). In vivo,
MEC-6 and MEC-4 colocalize in a punctate pattern along
the axons of touch neurons (51a). Chelur et al. (51a)
suggest that the punctate expression pattern resembles
the localization of membrane proteins that are associated
with lipid rafts and caveolae. They also suggest that
MEC-6 may function to restrict degenerin channel complexes to specialized membrane microdomains required
for mechanosensory transduction.
Figure 7 shows a working model of a mechanosensory ion channel complex in C. elegans touch neurons.
Mechanical force-induced displacement of the MEC-4/10
heteromultimer with respect to cytoskeletal and extracellular matrix attachment points is thought to gate the
channel open causing cation influx and membrane depolarization. In vivo measurement of mechanosensory channel activity has to date not been possible because of the
relative inaccessibility of touch neurons for patch-clamp
measurements. However, with the development of primary nematode cell culture methods (53), touch neurons
can now be patch clamped in vitro (54). Cultured touch
neurons have recently been shown to express mechanosensitive channel activity (55). The combination of patchclamp electrophysiology and genetics is likely to provide
unique insights into the molecular workings of mechanosensory neurons and mechanosensitive ion channels.
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
DRASIC gene increases the sensitivity of mechanoreceptors that detect light touch, but decreases the sensitivity
of receptors that respond to noxious pinch. Gene knockout also decreases the sensitivity of acid- and heat-sensitive nociceptors. These findings suggest that DRASIC
functions as part of mechanosensitive ion channel complexes as well as channel complexes that function in pain
perception.
B. Of Mice and Worms: New Insights Into ClC
Anion Channel Physiology
regulation of membrane potential, Cl⫺ transport in intracellular vesicles and epithelial Cl⫺ transport (104, 332).
Six ClC genes are present in the C. elegans genome
(229, 291). These genes have been termed clh-1 (Cl⫺
channel homolog) through clh-6 (229, 248) or CeClC-1
through CeClC-6 (291). I will use the clh nomenclature to
avoid confusing worm channels with their mammalian
counterparts.
Nematodes have a full complement of “mammaliantype” ClC channels. The six CLH channels are representative of the three mammalian ClC channel subfamilies.
GFP (29, 229, 291) and lacZ (248) reporter studies have
demonstrated that clh genes are expressed in a variety of
worm cell types including neurons, muscle cells, and
epithelial cells. CLH-4 expression may be restricted
largely to the excretory cell (229, 291) (e.g., Fig. 5).
Relatively little is known about the physiological
roles of C. elegans ClC channels. CLH-1 is expressed
primarily in the hypodermis. Null mutations increase
body width, which can be reversed by exposing animals
to hypertonic medium. These observations suggested to
Petalcorin et al. (248) that CLH-1 plays a role in whole
animal osmoregulation.
CLH-5 is a homolog of mammalian ClC-3, -4, and -5,
FIG. 8. CLH-3 anion channel activity in C. elegans oocytes. A: schematic diagram of the adult hermaphrodite gonad.
B: differential interference contrast micrographs of adult hermaphrodite gonad (left) and an isolated nonmaturing oocyte
(right). Oo, oocyte; Spt, spermatheca; Emb, embryo. Scale bar in both micrographs is 20 ␮m. C: activation of CLH-3
current in a C. elegans oocyte swollen by reducing bath osmolality. Whole cell currents were elicited by stepping
membrane voltage from ⫺100 to ⫹80 mV in 20-mV steps from a holding potential of 0 mV. D: whole cell anion currents
in a nonmaturing oocyte and an oocyte undergoing meiotic maturation. No CLH-3 activation is detected in the
nonmaturing oocyte. CLH-3 is active in the maturing oocyte and continues to activate for ⬃3 min after obtaining whole
cell access. [From Rutledge et al. (279), with permission from Elsevier Science.]
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Members of the ClC superfamily of voltage-gated Cl⫺
channels have been identified in plants, yeast, eubacteria,
archaebacteria, and various invertebrate and vertebrate
animals (104, 332). Although the function and regulation
of most ClC channels are obscure, the conservation of
ClC genes in widely divergent organisms suggests that
they play important and fundamental physiological roles.
Nine ClC genes have been described in mammals.
These genes comprise three distinct subfamilies: ClC-1, -2,
Ka/K1 and Kb/K2; ClC-3, -4, and -5; and ClC-6 and -7.
Knockout studies and the identification of disease-causing mutations indicate that ClC channels function in the
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tilization, and/or early development. However, RNAi
knockdown of channel expression also has no effect on
these reproductive events (279).
Oocytes are surrounded by smooth muscle-like myoepithelial sheath cells (see Fig. 8A). Before meiotic maturation, sheath cells contract weakly and intermittently at
a basal rate of 7– 8 contractions/min. Sheath cell ovulatory
contractions are triggered ⬃4 –5 min after the start of
meiotic maturation. During this contractile cycle, both the
force and rate of sheath contractions increase dramatically. At the peak of contractile activity, the spermatheca
dilates and the oocyte is ovulated into the spermatheca
where it is fertilized. This oocyte maturation and ovulation cycle is repeated every 20 – 40 min. Both sheath cell
ovulatory contractions and spermatheca dilation are controlled by signals from the maturing oocyte (207). The
precise mechanisms of intercellular signaling are incompletely understood.
Activation of CLH-3 in the maturing oocyte suggested
that the channel may function to regulate the ovulatory
process. Disruption of CLH-3 expression by RNAi had no
effect on the timing of spermatheca dilation and ovulation. However, knockdown of CLH-3 activity induced premature sheath cell ovulatory contractions. In clh-3
dsRNA-injected animals, sheath contractions began 1–2
min earlier than in control worms. These results suggest
that activation of CLH-3 in the maturing oocyte functions
as a negative or inhibitory regulator of sheath cell ovulatory contractions. This regulation may serve to tightly
synchronize sheath cell ovulatory contractions with meiotic cell cycle progression in the oocyte (279).
Reporter studies have failed to detect CLH-3 expression in sheath cells (229, 291). Rutledge et al. (279) therefore postulated that activation of CLH-3 in the maturing
oocyte modulates sheath cell contractions. Oocytes are
coupled to sheath cells by gap junctions (116), indicating
that the two cell types likely communicate by chemical
and electrical signals. Activation of CLH-3 may depolarize
the oocyte and electrically coupled sheath cell membranes. Depolarization in turn may inhibit Ca2⫹ influx into
the sheath cell via store-operated Ca2⫹ channels (279,
302). Recent studies have suggested that Ca2⫹ influx and
inositol 1,4,5-trisphosphate (IP3) signaling pathways play
important roles in regulating sheath cell contractility (X.
Yin, A. Broslat, E. Rutledge, C. Böhmer, and K. Strange,
unpublished observations).
CLH-3 and ClC-2 share 40% amino acid identity.
Given the conservation of their biophysical properties
and primary structure, we proposed that CLH-3 and ClC-2
are orthologs. Orthologs are genes in different species
that arose from a common ancestor and carry out similar
physiological roles (270). We suggested that like CLH-3,
ClC-2 may be regulated by cell cycle events, that it may
function in signaling and cell-to-cell communication path-
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which are thought to play important roles in endocytosis
and intracellular vesicle function (251, 301, 338). Consistent with this idea, knockdown of CLH-5 by RNAi reduces
endocytosis by coelomocytes (87), which are thought to
be scavenger cells that continuously endocytose fluid
from the pseudocoelom.
The most extensively studied C. elegans ClC channel
is CLH-3. As discussed in section IIC1C, nematode oocytes
are readily accessible for patch-clamp studies (see Fig. 8,
A and B). Under basal conditions, oocytes exhibit little
Cl⫺ channel activity. However, osmotic swelling causes
dramatic activation of an inwardly rectifying Cl⫺ current
(Fig. 8C). Strong hyperpolarization causes further current
activation. Detailed biophysical characterization of this
current demonstrated that the channel’s volume sensitivity, voltage sensitivity, anion selectivity, pharmacology,
and extracellular pH sensitivity are similar to those of
heterologously expressed mammalian ClC-2 as well as
native anion currents thought to be due to the activity of
ClC-2 (279). RNAi studies, single oocyte RT-PCR (279),
and heterologous expression (J. Denton, C. Böhmer, K.
Nehrke, and K. Strange, unpublished observations) demonstrated that the oocyte Cl⫺ channel is encoded by clh-3.
Further experimental analysis revealed an intriguing
pattern of CLH-3 activity. Oocytes must undergo DNA
reduction divisions or meiosis before fertilization. C. elegans oocytes are arrested in prophase I of meiosis I
during much of their development. Just before fertilization, meiosis is reinitiated, a process universally referred
to as meiotic maturation. In nonmaturing oocytes, CLH-3
is quiescent and can be activated by swelling. However, in
oocytes undergoing meiotic maturation, CLH-3 is constitutively active (Fig. 8D).
Pharmacological studies demonstrated that activation of CLH-3 during cell swelling and oocyte meiotic
maturation is controlled by serine/threonine dephosphorylation of the channel and/or accessory proteins
and that dephosphorylation is brought about by type 1
protein phosphatases (PP1). Consistent with these results, RNAi studies identified the CeGLC-7␣ and
CeGLC-7␤ phosphatases as those that mediate dephosphorylation and channel activation (280). These phosphatases share 85–93% amino acid identity with mammalian PP1␣ and PP1␤ and have been shown to play
important roles in regulation of C. elegans meiotic and
mitotic cell cycle events (136, 154, 277).
ClC-2 is widely expressed in mammalian cells and
tissues and has been proposed to function in transepithelial salt and water transport, intracellular Cl⫺ homeostasis, and cell volume regulation (104, 332). Disruption of CLH-3 expression by RNAi has no obvious
effect on the physiology of oocytes including their ability to volume regulate (279). Activation of the channel
during oocyte meiotic maturation suggests that CLH-3
may play a role in meiotic cell cycle progression, fer-
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
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C. Staying Regular: the IP3 Receptor
Rhythmic biological processes are found in all organisms ranging from bacteria to mammals (146, 166). These
rhythms are divided into three types: circadian, ultradian,
and infradian. Circadian and infradian rhythms have cycles of ⬃24 h (20 –28 h) and ⬎28 h, respectively. Ultradian
rhythms occur with periods ranging from milliseconds to
hours (⬍20 h) and play important roles in mammalian
physiology. Perhaps the most familiar ultradian rhythm is
the heartbeat.
Relatively little is known about the genes responsible
for ultradian timekeeping. However, C. elegans has provided important insights into this process. While they are
feeding, nematodes defecate once every 45–50 s or so
with little variation (192, 319). Defecation is mediated by
sequential contraction of the posterior body wall muscles,
anterior body wall muscles, and enteric muscles (Fig. 9A).
The defecation cycle is relatively insensitive to temperature change and has a phase that can be reset by external
stimuli such as gentle touch to the animal. In addition, the
phase of the rhythm is maintained even when defection
stops during periods that the animal is not feeding. The
characteristics of the defecation rhythm suggest that it is
controlled by an endogenous clock.
Iwasaki et al. (145) screened for mutant worms with
abnormal defection cycles. Twelve dec (defecation cycle
defective) genes were identified that when mutated cause
the cycle to slow down or speed up. The dec-4 gene
encodes an IP3 receptor (63). This gene is also termed
itr-1 (inositol trisphosphate receptor).
The itr-1 gene is the only IP3 receptor-encoding gene
in C. elegans. It is expressed widely in the organism
including the nervous system, excretory cell, gonad, pharynx, and intestine (23, 63). Three NH2-terminal variants
are encoded by itr-1 (23). Expression of the three isoforms generally occurs in nonoverlapping cell types and is
regulated by cell-specific promoters (110).
Mutations in itr-1 slow or eliminate the defecation
cycle, whereas overexpression of the gene increases defecation rate (63). Microinjection of fura 2 into intestinal
epithelial cells demonstrated that an intracellular Ca2⫹
spike occurs at the initiation of posterior body wall muscle contraction. Calcium spikes are slowed or absent in
animals with loss-of-function mutations in itr-1. Mutations in crt-1, which encodes the Ca2⫹-binding protein
calreticulin, causes further slowing of the defecation cycle in itr-1 mutants (240). This genetic interaction suggests that itr-1 and crt-1 function in Ca2⫹ signaling pathways that control defecation rhythm. Dal Santo et al. (63)
have proposed that IP3-dependent Ca2⫹ oscillations in
intestinal cells control the secretion of a signal that regulates contraction of the surrounding muscles that drive
defecation (Fig. 9B).
IP3 receptors are localized to the endoplasmic re-
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ways, and that it may regulate cell cycle-dependent physiological processes (279, 302).
Several lines of evidence support this hypothesis. In
ascidian embryos, a swelling- and hyperpolarization-activated inwardly rectifying Cl⫺ current with biophysical
properties similar to those of CLH-3 and ClC-2 is dramatically activated when cells enter M phase of the mitotic
cell cycle (31, 59). An inhibitor of cell cycle-dependent
kinases, 6-dimethylaminopurine, increases current amplitude suggesting that, like CLH-3, cell cycle-dependent
channel activation is mediated by protein dephosphorylation events (331).
Recently, Furukawa et al. (99) demonstrated that
rabbit ClC-2 is phosphorylated at serine-632 by the M
phase specific cyclin-dependent kinase (CDK) p34cdc2/
cyclin B. Serine-632 is located within a cyclin-dependent
kinase phosphorylation consensus site on the COOH terminus of the channel. Phosphorylation of serine-632 inhibits ClC-2 activity. PP1␣ and PP1␤ interact with the
COOH terminus of ClC-2 in yeast two-hybrid assays, suggesting that they may activate the channel by dephosphorylating serine-632 and/or other phosphorylation sites
(99). Recent studies of rat ClC-2 have shown that swelling-induced channel activation is triggered by serine/threonine dephosphorylation that is most likely mediated by
type 1 protein phosphatases (280).
Zheng et al. (363) have shown that levels of heterologously expressed ClC-2 protein are downregulated at the
completion of mitotic M phase. Furthermore, the channel
is ubiquitinated during M phase, and ubiquitination is
inhibited by inhibition of p34cdc2/cyclin B or by mutation
of serine-632 to alanine. Taken together, the studies in
ascidian embryos (31, 59, 331) and the findings of Furukawa et al. (99) and Zheng et al. (363) suggest that ClC-2
activity is regulated by cell cycle-dependent phosphorylation signaling pathways.
Recent studies in ClC-2 knockout mice also support
the hypothesis that CLH-3 and ClC-2 are orthologs. Loss
of ClC-2 function causes the retina and testes to degenerate progressively resulting in blindness and male sterility (34). What do the C. elegans hermaphrodite gonad and
mammalian testis and retina have in common? All three
organs contain cell types that interact closely with and are
functionally dependent on one another. In the mouse
testis and worm gonad, germ cell development and meiotic cell cycle events are controlled by heterologous cellcell interactions and intercellular communication (reviewed in Ref. 302). Bösl et al. (34) suggested that ClC-2 in
the mouse testis and retina functions to regulate localized
extracellular ionic environments that are essential for cell
function. It is also feasible that ClC-2 plays important
signaling roles in the testis and retina as we have proposed for the worm gonad (279, 302).
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ticulum (ER) where they function as IP3-activated Ca2⫹
release channels. Calcium released from the ER initially activates neighboring channels in a positive feedback loop termed calcium-induced calcium release. Increasing cytoplasmic Ca2⫹ levels has the opposite effect and inhibits channel activity (reviewed by Refs. 27,
239). Dal Santo et al. (63) propose two mechanisms by
which the IP3 receptor could function as a clock. First,
the timing of cytoplasmic Ca2⫹ spikes and thus the
Physiol Rev • VOL
defecation cycle could be critically dependent on the
time it takes for Ca2⫹ to diffuse to and activate neighboring channels. The stimulatory effect of overexpression of itr-1 on the defecation cycle is consistent with
this model. In addition, one of the itr-1 loss-of-function
alleles maps to a region adjacent to a Ca2⫹ binding site
identified in the mouse IP3 receptor (295). This mutation could disrupt cycle timing by decreasing the sensitivity of the channel to activating levels of Ca2⫹.
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FIG. 9. C. elegans defecation cycle. A: diagram illustrating muscle contractions that mediate defecation. Cycle is
initiated by contraction of posterior body wall muscles (pBoc). After relaxation of these muscles, the anterior body wall
muscles contract (aBoc) and then expulsion occurs by enteric muscle contraction (Emc). The cycle repeats itself every
45–50 s. [Based on Dal Santo et al. (63).] B: model illustrating possible role of intracellular Ca2⫹ in regulating defecation
cycle. Cyclical elevation of cytoplasmic Ca2⫹ levels driven in part by inositol 1,4,5-trisphosphate (IP3)-dependent release
of Ca2⫹ from intracellular stores triggers exocytic secretion from intestinal cells of an unidentified messenger that
induces pBoc (see Ref. 63). Execution of aBoc and Emc may be regulated in part by a neuronal circuit (209). PLC,
phospholipase C.
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
D. Cleaning House: ABC Transporters
The ATP-binding cassette (ABC) transporter superfamily is the largest transporter gene family. ABC transporters are responsible for the transport across plasma
and intracellular membranes of a wide variety of substrates such as metal ions, peptides, proteins, metabolites
and diverse hydrophobic compounds. The ABC transporters include multidrug resistance (MDR) and P-glycoprotein transporters, the cystic fibrosis transmembrane conductance regulator, and sulfonylurea receptors (67).
At least 61 genes in the C. elegans genome encode
predicted ABC transporters (http://www.biology.
ucsd.edu/⬃ipaulsen/transport/). Members of this gene
family are expressed in numerous cells and tissues including the intestine, excretory cell, pharynx, epithelial cells
of the vulva, and the germline (39, 40, 190, 352). Deletion
mutagenesis has demonstrated that ABC transporters
protect nematodes against heavy metals (39) and chemicals such as colchicine and chloroquine (40).
During somatic development, 1,090 cells are generated in a C. elegans hermaphrodite. Of these, 131 undergo
programmed cell death or apoptosis primarily during embryonic development and to a lesser extent during the L2
stage. As with other aspects of development, programmed
cell death is largely invariant (307, 308). This characteristic allows the study of cell death at the single-cell level
and greatly facilitates identification of genes involved in
the process. Indeed, much of the understanding of apoptosis in mammalian cells has been foreshadowed by
studies in C. elegans.
Apoptotic cells undergo a series of characteristic
morphological changes that include a decrease in refractivity and texture of the cytoplasm and an increase in
refractivity of the nucleus. Subsequently, the cells appear
as round, flat disks and refractivity of the cytoplasm
increases. Neighboring cells gradually engulf cells with a
flat, disk-shaped appearance (127).
Physiol Rev • VOL
The ced-7 gene encodes a putative ABC transporter
that is expressed widely in the developing embryo and is
localized to plasma membranes (352). Mutations in ced-7
prevent phagocytic engulfment of apoptotic cells, and its
function is required in both the engulfing cells and cells
undergoing apoptosis (83, 352). Zhou et al. (364) have
suggested that CED-7 may function in the transport of
adhesion molecules that facilitate physical contact between phagocytic and dying cells and/or in the transport
of a “cell corpse signal” onto the cell surface. CED-7 may
also regulate the function of membrane receptors required for phagocytosis. Interestingly, ABC1 is a mammalian structural ortholog of CED-7 (352). ABC1 is involved
in the phagocytosis of apoptotic cells by macrophages,
transbilayer redistribution of phosphatidlyserine, and export of cholesterol and phospholipids from cells (119).
Phospholipid signals likely play important roles in regulating the engulfment process (128).
E. Life and Death and Things in Between: TRP and
Related Channels
The ability to perform detailed physiological and genetic analyses has made Drosophila a powerful model
system for defining the molecular mechanisms of phototransduction. A simple extracellular recording technique,
the electroretinogram (ERG), is used to measure the
summed light response of all cells in the fly retina. Exposure of the Drosophila retina to light induces a sustained
light-coincident receptor potential (LCRP). However, in
certain Drosophila mutants, the receptor potential is transient. Identification and heterologous expression of the
transient receptor potential (trp) gene demonstrated that
it encodes a Ca2⫹-permeable cation channel activated by
a phospholipase C-mediated signaling cascade (137, 223,
224, 246, 249, 327).
Following these initial studies in Drosophila, an explosion of research activity has identified numerous trp
genes in vertebrate and invertebrate animals. In mammals, trp genes encode a family of at least 20 ion channelforming proteins with six transmembrane segments. TRP
channels are cation channels that mediate Ca2⫹ influx
into cells. Three subfamilies of TRP channels were identified originally. Harteneck et al. (123) termed these
subfamilies “short,” “osm-9-like,” and “long.” The nomenclature of these subfamilies has now been changed to
TRP-canonical (TRPC), TRP-vanilloid (TRPV), and TRPmelastatin (TRPM), respectively (222). Three additional
subfamilies have also been identified. These are termed
TRP-NOMPC (TRPN), TRP-polycystin (TRPP), and TRPmucolipidin (TRPML) (221).
At least 14 TRP-encoding genes are present in the C.
elegans genome. The physiological functions and regulation of most TRP channels are incompletely understood,
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Second, cycle timing could be affected by the time it
takes channels to recover from inactivation induced by
high Ca2⫹ levels. Mutations that prolong recovery
would slow cycle time.
Mutations in other genes including calmodulin kinase
II (267) and a DEG/ENaC channel that is expressed in the
intestinal cells (313) also disrupt the defecation cycle.
Ultradian timekeeping in C. elegans thus involves interaction between IP3-regulated Ca2⫹ signaling pathways,
protein phosphorylation events, and ion channels that
likely regulate intestinal cell membrane potential. Thus
studying something as seemingly obscure and simple as
worm defecation is likely to ultimately provide a detailed,
integrated understanding of the molecular workings of
oscillatory Ca2⫹ signals.
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KEVIN STRANGE
but many of them play important roles in sensory transduction. TRP channels have also been proposed to function as store-operated Ca2⫹ influx channels and to play
roles in epithelial salt and water transport (221).
1. Life and death: gon-2 and ced-11
Physiol Rev • VOL
2. Avoiding stress: osm-9 and ocr-2
Nematodes sense the external environment in part
through a pair of openings in the cuticle on their head
termed amphids. Eight neurons associate directly with
each amphid pore and contact the external environment
via dendrites that terminate in sensory cilia. Another four
sensory neurons have dendrites that associate with a
support cell termed the amphid sheath cell. Axons extend
from the cell bodies of the amphid neurons to the nerve
ring where they make synaptic contacts with other neurons. Laser ablation studies have demonstrated that the
amphid neurons function in thermosensation, chemosensation, and mechanosensation (19).
C. elegans is attracted to low concentrations of salts,
sugars, and other chemicals. However, at high concentrations, worms initiate an avoidance response to these substances (340). Culotti and Russell (62) suggested that the
repellent effect is due to the high osmolality of solutions
of these substances and identified mutant worm strains
that are defective in osmotic avoidance behavior.
osm-9 (osmotic avoidance defective) mutants are
defective in their ability to detect hypertonic media, nose
touch, volatile repellents, and chemical attractants (57,
58). Chemotaxis is mediated by AWA amphid neurons,
and ASH amphid neurons mediate the avoidance responses to nose touch, volatile repellents, and hypertonic
media (19). GFP reporter studies demonstrated that
OSM-9 is expressed in both neuron types and is localized
to sensory cilia in AWA (58).
The osm-9 gene encodes a 937-amino acid protein
with substantial homology to TRP channels (58) and represents the first identified member of the TRPV subfamily.
Behavioral data suggest that the OSM-9 channel functions
as part of sensory protein complexes that detect chemicals or odorants, hypertonic media, and mechanical force.
Because the sensory cilia of ASH neurons are in direct
contact with the external environment, it seems likely
that OSM-9 detects hypertonicity-induced cell shrinkage.
Unfortunately, it has not yet been possible to heterologously express osm-9 and characterize channel functional
properties and regulation (321a; X. Yin and K. Strange,
unpublished observations). However, consistent with the
role of osm-9 in osmosensation and mechanosensation, a
mammalian TRPV channel has recently been shown to be
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C. elegans is small and transparent, has a relatively
simple anatomy, and develops rapidly with a largely invariant cell lineage (307, 308). These characteristics combined with its genetic tractability have made this organism a particularly valuable model system in which to
identify genes that control the birth and death of cells.
Studies of gonadogenesis in C. elegans have identified a number of genes important for cell growth and
proliferation (140). The gonad primordium of newly
hatched L1 larvae consists of two somatic cells, Z1 and Z4,
and two germs cells, Z2 and Z3. Z1 and Z4 divide throughout larval development giving rise ultimately to the gonad
distal tip and sheath, the spermatheca and the uterus.
Initiation and continuation of Z1 and Z4 mitotic divisions
require the activity of gon-2 (gonadogenesis abnormal)
(309). Loss-of-function mutations in gon-2 lead to delayed, arrested, or incomplete cell divisions. The gon-2
gene encodes a putative TRPM channel (345) that may
mediate Ca2⫹ influx in response to extracellular regulatory signals. Calcium signaling pathways have been
shown to play critical roles in controlling cell division in
numerous organisms (346).
As discussed in section IIID, a number of ced (abnormal programmed cell death) genes have been identified
by characterizing apoptosis in mutagenized worms. The
ced-11 gene encodes a putative TRPM channel. The timing of apoptotic cell death and engulfment are not altered
in ced-11 mutants, but dying cells have abnormal morphology (G. Stanfield and H. R. Horvitz, personal communication).
It is well known that extreme increases in cytoplasmic Ca2⫹ levels play a role in triggering both necrotic and
apoptotic cell death (357). Accumulating evidence also
suggests that IP3-regulated intracellular Ca2⫹ signaling
pathways play a role in inducing apoptosis (114). As noted
earlier, TRP channels have been proposed to function as
store-operated Ca2⫹ influx pathways (221). It is therefore
reasonable to postulate that CED-11 may function in Ca2⫹
signaling pathways that regulate aspects of programmed
cell death in C. elegans.
The mammalian melastatin gene has been shown
recently to encode a Ca2⫹-permeable TRP channel (353).
Interestingly, melastatin expression levels are inversely
correlated with the metastatic potential of human melanomas (76, 77). TRP-p8 encodes a putative TRPM channel
that is expressed primarily in prostate epithelial cells.
Expression levels are increased in prostate tumors as well
as in nonprostatic primary tumors of breast, colon, lung,
and skin (323). MTR1 is also a member of the TRPM
family and maps to a chromosome region associated with
Beckwith-Wiederman syndrome, a disease that predisposes individuals to neoplasias (84, 256). The phenotypes
of worms expressing mutations in gon-2 or ced-11 and the
possible involvement of melastatin, TRP-p8 and MTR1 in
tumorogenesis suggests that TRP channels may play
widespread roles in controlling cell cycle events, cell
growth, and programmed cell death.
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
3. Sex and polycystic kidney disease: lov-1 and pkd-2
Autosomal dominant polycystic kidney disease is
characterized by progressive development of epithelial
cysts in the kidney, liver, and pancreas (299). The genes
defective in the disease are polycystin-1 (PKD-1) and
polycytstin-2 (PKD-2). PKD-1 is a ⬃4,300 amino acid
membrane glycoprotein with 11 predicted transmembrane domains and a very large extracellular NH2 terminus. The predicted structure suggests that PKD-1 functions in cell-cell interactions or in interactions with the
extracellular matrix (141).
PKD-2 is a 968 amino acid membrane protein with 6
predicted transmembrane domains (218). The protein
Physiol Rev • VOL
shares significant homology with TRP channels (324).
Consistent with this finding, PKD-2 has been shown to
interact with TRPC1 (324), and PKD-1 and PKD-2 interact
to form Ca2⫹-permeable cation channels when expressed
heterologously in Chinese hamster ovary cells (120).
PKD-2 is also capable of forming Ca2⫹-permeable cation
channels when expressed alone (52, 329) or reconstituted
in lipid bilayers (107). Recent studies by Koulen et al.
(174) suggest that PKD-2 may function as a Ca2⫹-activated
intracellular Ca2⫹ release channel.
Nematodes possess both PKD-1 and PKD-2 homologs
termed LOV-1 (location of vulva) and PKD-2, respectively
(21). Mutations in lov-1 and pkd-2 disrupt the male mating
behaviors, “response” and “vulva location.” When a male
worm encounters a hermaphrodite, he responds by placing his tail against her body and moving backwards. Upon
reaching the end of the hermaphrodite body, the male
turns and continues backing until he locates the vulva.
Mating occurs by insertion of the copulatory spicules into
the vulva, which is followed by ejaculation (194).
Response and vulva location are mediated by nine
pairs of sensory rays and a cuticular structure called the
hook located on the male tail (194). The lov-1 and pkd-2
are coexpressed in male sensory neurons that innervate
the rays and hook (20, 21), and they may interact genetically (20). Barr et al. (20) have suggested that LOV-1
functions as a chemosensory or mechansensory receptor
that detects mechanical and/or chemical signals from the
hermaphrodite during mating behavior. These signals may
in turn regulate PKD-2 cation channel activity.
4. Being sensitive: feeling bacteria with Ce-nompC
C. elegans slows its locomotory rate when it comes
into contact with bacteria (290). This behavior is thought
to be an adaptive mechanism that allows the animals to
spend more time in contact with a food source. The basal
slowing response is lost when the animal’s eight dopaminergic neurons are destroyed by laser ablation. These
dopaminergic neurons include four CEPs, two ADEs, and
two PDEs. Ciliated dendrites from these neurons are
embedded in the cuticle of the nose and lateral body wall,
suggesting that they have a mechanosensory function
(348).
Worms also slow their movement when they crawl
through sterile 20- to 50-␮m-diameter Sephadex beads
(290). These beads are much larger than bacteria and
cannot be ingested by the worms. The Sephadex-induced
slowing response is also lost when the dopaminergic neurons are laser ablated. These findings suggest that the
ciliated dendrites of dopaminergic neurons detect a mechanical signal from bacteria.
How does a sensory neuron feel bacteria? Recent
studies by Zucker and co-workers (337) in Drosophila
have shed light on this problem. Fruit flies can hear, sense
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activated by hypotonic stress when expressed heterologously (188, 304). This suggests that some TRP channels
may sense cell volume changes, possibly via mechanical
forces transmitted directly through the lipid bilayer
and/or cytoskeletal attachments.
Osmotic avoidance and response to nose touch are
also dependent on another member of the TRPV subfamily, OCR-2 (osm-9/capsaicin receptor related) (321a).
OCR-2 is localized to sensory cilia similar to OSM-9
(321a). Interestingly, coexpression of the two proteins
may be required for subcellular compartmentation and
cilia localization (321a). These results suggest that OSM-9
and OCR-2 are components of a heteromeric channel
and/or they function in a multiprotein signaling complex.
Recently, OSM-9 function has been linked to social
behavior in C. elegans (67b). Natural strains of C. elegans
are either solitary feeders or they aggregate and feed in
groups. Variation of a single amino acid in a putative
neuropeptide Y-like receptor (NPR-1) is responsible for
this behavioral difference (67a). Mutations in osm-9 and
ocr-2 abolish aggregation and group feeding. Simultaneous laser ablation of both pairs of ASH and ADL neurons also abolishes group feeding (67b). Like ASH neurons, ADL neurons mediate avoidance responses to noxious stimuli.
de Bono et al. (67b) suggest that ASH and ADL neurons transmit stressful and repulsive environmental signals to a neuronal circuit that induces aggregation and
group feeding. The function of these neurons requires
activity of the putative OSM-9/OCR-2 channel. de Bono et
al. (67b) make the intriguing proposal that group feeding
functions as a defense mechanism in C. elegans under
environmentally stressful conditions. Aggregation may allow more concentrated secretion of enzymes that inactivate toxins produced by bacteria and other organisms.
Similarly, aggregation would allow concentrated secretion of the pheromone that promotes development of
dauer larvae (243, 297). As noted in section IIB1, dauer
larvae aid in survival under stressful conditions and in the
dispersal of C. elegans to new habitats (243, 297).
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F. Getting the Message Out: Neurotransmitter
Transporters and Ligand-gated Channels
The relative anatomical simplicity of the C. elegans
nervous system (see sect. IIB3C) has greatly facilitated the
study of a variety of fundamental neurobiological problems such as neuronal development and neural control of
behavior. C. elegans has also provided a powerful model
system for understanding the signaling roles and transport of neurotransmitters. The complement and functions
of neurotransmitters utilized by nematodes are similar to
those of vertebrates (263), and identification of nematode
neurotransmitter transporters through forward genetic
screens has directly aided the cloning and characterization of mammalian homologs (e.g., Refs. 25, 278, 312).
1. The art and science of a genetic screen:
DAT and VGAT
Three catecholamine neurotransmitters, dopamine,
serotonin, and octopamine, have been identified in C.
Physiol Rev • VOL
elegans (263). DAT-1 is a plasma membrane Na⫹ and
Cl⫺-dependent dopamine transporter expressed in the
eight dopaminergic neurons of C. elegans (149, 227). Dopamine transporters play a critical role in dopamine signaling by regulating the extracellular levels of this neurotransmitter. In mammals, DATs are also important targets
of psychostimulants and provide a transport pathway for
the entry of neurotxoins into cells (6, 215).
Nass et al. (227) demonstrated recently that the neurotoxin 6-hydroxydopamine (6-OHDA) induces dopaminergic neuron degeneration in C. elegans. Degeneration is
blocked completely by knockout of the dat-1 gene, demonstrating that transport of 6-OHDA into the cells is required for its toxic effects. Dopaminergic neurons were
identified in these studies using a dat-1::GFP reporter
(Fig. 5A). When worms are exposed to 6-OHDA, the GFP
fluorescence disappears as the neurons degenerate. Destruction of the dopaminergic neurons has no effect on
worm viability. Loss of GFP can be detected readily by
eye using a stereo dissecting microscope equipped for
fluorescence imaging. It may also be possible to automate
GFP detection using technology based on fluorescenceactivated cell sorting (98).
The ability to assess toxicity using a stereo dissecting
microscope provides the basis for a straightforward and
rapid screening assay. GFP-labeled dopaminergic neurons
remain intact in mutagenized worms that are resistant to
6-OHDA (227). Identification of the genes conferring resistance will likely provide unique insights into the molecular basis of DAT-1 regulation, trafficking and localization, and neurotoxin-induced cell death (228). Knowledge
of the genetics of DAT-1 function has important implications not only for understanding solute transporter function and regulation, but also for understanding and treating addiction and neurodegenerative disorders such as
Parkinson’s disease.
McIntire et al. (210) employed a genetic strategy to
identify C. elegans VGAT, a vesicular GABA transporter
that represented the first member of a new neurotransmitter transporter family. These investigators reasoned
that worms with defective vesicular GABA transport
would exhibit behavioral defects similar to those of
worms in which GABAergic neurons had been laser ablated. Destruction of the 26 GABAergic neurons causes
defects in locomotion, defecation, and head movement
during foraging (209). Mutations in several genes were
also known to cause these defects (208, 268). However,
mutation of only one gene, unc-47, generated a phenotype most consistent with disruption of vesicular GABA
transport. The unc-47 mutations are presynaptic, affect
all behaviors controlled by GABA, and cause GABA to
accumulate in GABAergic neurons. Identification and characterization of unc-47 demonstrated that the gene encodes
a membrane protein associated with synaptic vesicles.
Heterologous expression of the rat homolog of UNC-47
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touch, and detect body position and movement. Mechanical force is detected through a variety of mechanoreceptors including sensory bristles that consist of hollow hair
shafts innervated with sensory neurons.
Mechanoreceptor currents (MRCs) can be measured
in the bristles by placing a patch pipette over the cut end.
Directional displacement of the bristle by as little as 100
nm, which displaces the associated sensory neuron by an
estimated 2 nm, triggers an MRC with a latency of ⬃200
␮s (337). Forward genetic screens identified a gene
termed nompC (no mechanoreceptor potential) that is
essential for the function of mechanosensory bristle organs and fly proprioception. Mutations in nompC cause
flies to become uncoordinated and have abnormal MRCs.
nompC encodes a putative mechanosensory ion channel
forming protein with six predicted transmembrane segments. The NH2 terminus is 1,150 amino acids in length
and contains 29 ankyrin repeats, which is the largest
number found in any known protein. The transmembrane
segments share low but significant homology with TRP
channels.
A homologous gene, Ce-nompC, is present in the C.
elegans genome. The predicted protein shares ⬃40%
amino acid identity with Drosophila NOMPC including
the presence of 29 NH2-terminal ankyrin repeats. CeNOMPC is expressed in the ciliated dendrites of CEP
and ADE neurons (337). Taken together with the studies of Sawin et al. (290), these findings suggest that
mechanical distortions of the worm cuticle induced by
bacteria displace CEP and ADE sensory cilia. This displacement in turn activates Ce-nompC and triggers an
action potential that ultimately signals the worm that
food is present and that it should slow down to investigate.
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
induces GABA transport with functional properties similar to those observed in isolated synaptic vesicles.
2. Changing roles: from plasma membrane phosphate
transporter to VGLUT
Physiol Rev • VOL
It is uncertain whether this represents an important physiological function of the transporter or whether it is an
artifact of heterologous expression.
3. Foraging for food: the role of ligand-gated
ion channels
As discussed in section IIIE4, nematodes slow their
locomotion when a bacterial food source is encountered.
Slowing is mediated by the neurotransmitter serotonin
(290). When deprived of food for brief periods of time, the
slowing response is enhanced (264). Horvitz and co-workers (264) have screened for mutants defective in this
enhanced slowing. The mod-1 (modulation of locomotion
defective) mutants show significantly less slowing in the
presence of food than do wild-type animals (264). In
addition, mod-1 mutants are largely unaffected by exposure to exogenous serotonin, which immobilizes wildtype worms. Cloning of mod-1 and expression in Xenopus
oocytes demonstrated that the gene encodes a novel serotonin-gated Cl⫺ channel. Serotonin-gated Cl⫺ currents
have been detected in other invertebrate organisms (e.g.,
Ref. 185). However, it remains to be determined whether
mammalian homologs of MOD-1 exist.
Brockie et al. (38) cloned a full-length C. elegans
cDNA that encodes a predicted N-methyl-D-aspartate
(NMDA) receptor, NMR-1. NMR-1 is expressed at synapses of interneurons of the worm locomotory circuit.
Patch-clamp measurements demonstrated that NMDAsensitive currents in interneurons are mediated by
NMR-1. nmr-1 deletion mutants exhibit no gross defects
in muscle contraction, locomotion, and sensory behavior.
However, more detailed analysis of worm behavior revealed subtle defects. During foraging, wild-type worms
move forward for ⬃25 s, stop and move backwards
briefly, and then move forward again in a new direction.
This behavior allows the worm to correct errors in trajectory toward a presumptive food source. The nmr-1
deletion mutants alter the direction of their forward
movement less frequently and exhibit a reduced ability to
navigate a maze toward a chemotactic target. Expression
of a nondesensitizing variant of GLR-1 in the interneurons
rescues the nmr-1 deletion phenotype. These findings
suggest that long-lived NMR-1 currents function to amplify or integrate sensory information that regulates backward movement and direction changes required for efficient foraging.
4. Chloride channels and antibiotic resistance
Avermectins are a widely used class of anthelmintic
drugs that activate glutamate-gated Cl⫺ channels (69, 70,
245, 328). Screens for avermectin sensitivity in C. elegans
have identified avr (avermectin resistance abnormal) mutants. Mutations in avr-15 cause defects in pharyngeal
pumping (69). M3 IPSPs are not detected in EPG (see
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Screens for defects in pharyngeal pumping have identified eat (eating abnormal) mutants in C. elegans. The
normal pharyngeal pumping cycle consists of rhythmic
contraction and relaxation of the pharyngeal muscle. Contraction draws liquid and suspended bacteria into the
pharynx. Relaxation expels the fluid through the worm’s
mouth while the bacteria are retained (11). The relaxation
phase is regulated by M3 motor neurons that synapse with
the pharynx and generate inhibitory postsynaptic potentials
(IPSPs; see Fig. 3D) in the contracted muscle (10, 260).
Pharyngeal muscle relaxation is greatly delayed and
M3 motor neuron IPSPs are virtually absent in eat-4 mutants (11, 182, 260). Isolated eat-4 pharynxes show a
normal response to glutamate, suggesting that the defect
in M3 neurotransmission is presynaptic (69). The eat-4
mutants also exhibit defects in other functions controlled
by glutamatergic neurotransmission including responses
to nose touch, hypertonic solutions, and volatile odorants
(26) as well as in habituation, a simple form of learning
(265). These findings suggested that mutations in eat-4
reduce glutamate release into synaptic junctions.
The eat-4 gene is expressed in C. elegans glutamatergic neurons and encodes a membrane protein with
significant homology to the brain-specific, sodium-dependent phosphate transporter BNPI (182). Lee et al. (182)
suggested that EAT-4 functions as a phosphate transporter and that phosphate transported into the neuron
controls glutamate synthesis by regulating a phosphateactivated glutaminase that hydrolyzes glutamine to glutamate.
Characterization of the eat-4 mutant phenotype and
identification of the eat-4 gene provides an interesting
example of how insights gained from a model organism
can provide important clues into mammalian physiology.
When first cloned, BNP1 was proposed to play a role in
regulating cytoplasmic phosphate levels in neurons (233).
Immunolocalization studies in rat brain stimulated in part
by the demonstrated role of eat-4 in glutamate neurotransmission demonstrated that BNPI was mainly associated with synaptic vesicles of glutamatergic neurons (24).
Subsequent heterologous expression studies demonstrated that BNPI transports glutamate and that the functional characteristics of the transporter are the same as
those observed for glutamate transport in brain synaptic
vesicles (25, 312). EAT-4 is likely carrying out a similar
role in C. elegans. An intriguing question about BNPI that
remains is that the protein is also capable of transporting
phosphate into cells in a Na⫹-dependent manner when
expressed heterologously in the plasma membrane (233).
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KEVIN STRANGE
would thus require that resistance mutations occur simultaneously and therefore more rarely in multiple genes.
G. Complex Simplicity: Kⴙ Channels
More than 70 genes encode K⫹ channels in C. elegans. These channels fall into three major classes: six
transmembrane (TM) domain voltage-regulated K⫹ channels, two TM inward rectifiers, and four TM TWK channels that have two pore-forming domains per subunit (17,
344). Approximately half the worm’s K⫹ channel genes
encode TWK channels. The number of TWK channels and
their diversity are substantially greater than in Drosophila
or humans. Salkoff et al. (284) suggest that TWK channels,
which function as “leak” conductances regulated by diverse factors such as pH, temperature, membrane stretch,
and neurotransmitters (184), may serve to “fine tune” the
function of individual neurons and allow C. elegans to
optimize the functionality of the nervous system’s limited
physical circuitry.
A number of C. elegans K⫹ channels including high
conductance Ca2⫹-activated K⫹ channels, voltage-gated
K⫹ channels, KQT K⫹ channels, and eag-like K⫹ channels
are conserved in higher species, suggesting that they
arose in a common metazoan ancestor. C. elegans pro-
⫹
FIG. 10. Role of EXP-2 K channel in regulating excitability of C. elegans pharyngeal muscle. A: action potentials in
pharyngeal muscles from wild-type, exp-2 gain-of-function heterozygotes [exp-2(gf)/⫹] and exp-2 null [exp-2(0)] worms.
B: effect of gain-of-function mutation on EXP-2 K⫹ channel activity. The gain-of-function exp-2 allele is a cysteine-totyrosine mutation at amino acid 480 (C480Y) in the S6 domain of the channel. Wild-type EXP-2 expressed in Xenopus
oocytes exhibits slow depolarization-induced activation and very rapid inactivation. Recovery from inactivation upon
membrane repolarization is voltage dependent and also very rapid. EXP-2 C480Y is constitutively active even at strongly
hyperpolarized membrane potentials. Coexpression of wild type and C480Y EXP-2 in a 1:1 ratio, which may mimic
channel composition in exp-2 gain-of-function heterozygotes [exp-2(gf)/⫹], forms channels that are active at more
negative membrane potentials. Action potentials (see A) and hence pharyngeal pumping are greatly shortened in exp-2
gain-of-function heterozygotes, presumably because EXP-2 is activate at more negative membrane potentials. Loss of
EXP-2 activity greatly prolongs pharyngeal action potentials and pumping. [From Davis et al. (64), copyright 1999
American Association for the Advancement of Science.]
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sect. IIC1B and Fig. 3D) of avr-15 mutants, and the pharyngeal contraction cycle is greatly prolonged. The pharynx of these animals is also insensitive to iontophoretically applied glutamate. In wild-type animals, glutamate
hyperpolarizes the pharynx and shortens the duration of
muscle contraction during pumping. This effect is observed in pharynxes isolated from animals in which the
M3 motor neurons are laser ablated, demonstrating that
glutamate acts directly on the pharyngeal muscle. Cloning
of the avr-15 gene demonstrated that it encodes GluCl␣2,
a glutamate-gated Cl⫺ channel ␣-subunit expressed in the
pharynx. Taken together, these studies indicate that glutamate released from M3 neurons inhibits contraction by
activating pharyngeal glutamate-gated Cl⫺ channels that
hyperpolarize the muscle.
The avr-14 and glc-1 genes also encode ␣-subunits of
glutamate-gated Cl⫺ channels that appear to be expressed
extrapharyngeally (70). Interestingly, simultaneous mutation of glc-1, avr-14, and avr-15 is required to confer
significant resistance in C. elegans to the anthelmintic
drug ivermectin. Mutation of only two of these channels
confers modest or no resistance. These results suggest
that an important principle in the design and use of antibiotic drugs may be to target them to members of multigene families. Development of resistance to the drug
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
vides a tractable model system in which to define the
integrative physiology of these channel types.
1. A pump is a pump: exp-2 and regulation
of cell excitability
Physiol Rev • VOL
This constitutive channel activity likely explains why
C480Y homozygotes do not eat and die shortly after hatching (64). Coexpression of wild-type and C480Y EXP-2 in a
1:1 ratio, which may mimic channel composition in C480Y
heterozygotes, forms channels that are actived at more
negative membrane potentials (64) (Fig. 10B).
The kinetic properties of EXP-2 give rise to strong
inward rectification and are distinct from those of other
Kv-type channels. The channel functionally resembles
HERG (human ether-a-go go related gene) K⫹ channels
(91, 254). EXP-2 plays a similar physiological role to
HERG in the vertebrate heart. Both channels are inactivated during the plateau phase of the action potential but
recover from inactivation rapidly generating large tail
currents and contributing significantly to membrane repolarization. This is well illustrated by pharyngeal action
potentials observed in wild-type and mutant worms (Fig.
10A). Action potentials and hence pharyngeal pumping
are greatly shortened in C480Y heterozygotes, presumably
because EXP-2 activates at more negative membrane potentials. In EXP-2 null mutants, pharyngeal action potentials and pumping are greatly prolonged. Similarly, certain
mutations in HERG cause a form of long Q-T syndrome,
LQT2, which is a prolongation of the cardiac action potential (148).
Although the kinetic properties of EXP-2 are similar
to those of HERG, the mechanisms of fast inactivation are
different, and the channels have very different unitary
conductances (91). In addition, the channels are structurally unrelated. This indicates that the kinetic properties of
EXP-2 and HERG channels evolved independently as a
solution to a common physiological problem, namely, the
maintenance and regulation of long-duration action potentials and rhythmic electrical and contractile activity.
2. SLOing down neurotransmitter release
with Ca2⫹-activated K⫹ channels
The unc-64 gene encodes syntaxin (235, 283), a protein that plays an integral role in the fusion of neurotransmitter vesicles with the plasma membrane (189). Mutations in unc-64 cause various degrees of paralysis and
uncoordinated movement in C. elegans (235, 283). Wang
et al. (339) mutageneized unc-64 mutant worms and
screened for genes that suppress the paralyzed phenotype. This screen identified a number of genes that are
known to be important in regulating neurotransmitter
release. One novel gene identified in the screen was slo-1.
The slo-1 gene encodes a protein highly homologous
to high-conductance Ca2⫹-activated K⫹ channels that
were first identified in the Drosophila mutant slowpoke
(9, 82). Expression of slo-1 in Xenopus oocytes induces
voltage- and Ca2⫹-activated K⫹ currents. Suppressing alleles of slo-1 are the result of loss-of-function mutations
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The pharynx of C. elegans is a relatively simple neuromuscular pump that shares some developmental properties with the vertebrate heart (125), and pharyngeal
action potentials resemble those in cardiac muscle. Genetic characterization of pharyngeal contractile activity
has provided important insights into the molecular basis
of cellular excitability. This point is illustrated by a series
of studies carried out recently by Avery and co-workers
(64, 86, 91).
The exp-2 (expulsion step of defecation abnormal)
gene was identified originally in screens for animals with
defects in defecation behavior (319). exp-2 mutants also
exhibit defects in pharyngeal pumping. Animals with a
gain-of-function mutation in exp-2 have pharyngeal contractions that are faster and shallower than wild-type
worms. exp-2 null mutants have greatly prolonged pharyngeal contractions.
Figure 10A illustrates the time course of action potential generation in wild-type animals and exp-2 mutants.
The wild-type action potential shows a rapid depolarization of 60 –75 mV from a resting membrane potential of
about ⫺40 mV. Depolarization is followed by a slowly
declining plateau phase lasting 150 –250 ms, a rapid repolarization to ⫺70 mV, and then a slow depolarization back
to resting membrane potential. Membrane depolarization
and repolarization are largely normal in exp-2 gain-offunction heterozygotes [exp-2(gf)/⫹]. However, the plateau phase of depolarization is greatly shortened, and
resting membrane potential is hyperpolarized by about
⫺30 mV. In exp-2 null mutants [exp-2(0)], resting membrane potential and depolarization are normal, but the
plateau phase is greatly prolonged, and there is little
overshoot of membrane potential during repolarization
(64).
The exp-2 gene encodes a six transmembrane domain, voltage-activated K⫹ channel (64). The channel is
homologous to Kv-type channels and is expressed in pharyngeal muscle. The gain-of-function exp-2 allele is a cysteine-to-tyrosine mutation at amino acid 480 (C480Y) in
the S6 domain. This mutation is close to amino acid
positions shown to be important in regulating gating in
other Kv-type channels (135, 196, 238).
When expressed in Xenopus oocytes, wild-type
EXP-2 exhibits slow depolarization-induced activation
and very rapid inactivation. Recovery from inactivation
upon membrane repolarization is voltage dependent and
also very rapid (64, 86, 91) (Fig. 10B). Expression of
EXP-2 C480Y gives rise to channels that are active even at
strongly hyperpolarized membrane potentials (Fig. 10B).
405
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KEVIN STRANGE
Physiol Rev • VOL
results indicate that loss of SLO-1 function increases neurotransmitter release.
The findings on C. elegans SLO-1 add to a growing
body of literature indicating that Ca2⫹-activated K⫹ channels play a key role in regulating synaptic function. SLOtype K⫹ channels have been shown to colocalize with
voltage-gated Ca2⫹ channels in synaptic membranes, and
their activity appears to be tightly coupled (144, 274, 276,
356). Calcium influx through voltage-gated Ca2⫹ channels
activates neighboring SLO-type channels, which in turn
function to repolarize the membrane thereby reducing
Ca2⫹ influx and neurotransmitter release. The enhanced
neurosecretion and neurotransmission observed in C. elegans slo-1 loss-of-function mutants illustrate this emerging physiological principal in a compelling way.
H. Conclusions and Future Perspective
Genome sequences are often referred to as “molecular” or “genetic blueprints.” Blueprints are detailed, integrated sets of plans or programs of action that describe
how to accomplish a particular task. Given our current
level of understanding, a genome is little more than lines
of code (i.e., genes) that specify how to synthesize proteins. Genome sequence must be deciphered into a set of
instructions that allow us to understand when proteins
are synthesized, when and how groups of proteins are
assembled into functional complexes and pathways that
accomplish specific tasks, and when and how tasks are
assembled to build and run organelles, cells, tissues and
organs, and the whole organism. We are very far indeed
from having a molecular blueprint for even the simplest of
organisms much less a human.
It is the job of the integrative physiologist to decipher
the lines of an organism’s genetic code into a working
blueprint. Enormous intellectual and technical challenges
must be overcome to achieve this goal. The experimental
strategies that will be brought to bear on this problem
range from single gene and protein structure/function
studies to high-throughput genome-scale screens. Importantly, to learn how to build and run a human, we must
first learn how to build and run less complex organisms.
Genetically tractable organisms such as C. elegans
provide numerous experimental advantages for defining
gene function and integrative biology. While some mammalian physiologists may question the value of studying
bacteria, yeast, nematodes, and fruit flies, the inescapable
conclusion that we have learned from these and other
model organisms is that biology is rarely reinvented by
natural selection. Instead, natural selection takes a working plan and modifies it. Addressing complex physiological problems in a less complex organism that is genomically defined, genetically tractable and where it is more
straightforward and economical to manipulate gene func-
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that cause premature termination of the protein or translation of nonfunctional channels (339).
The slo-1 mutations by themselves have little effect
on worm locomotion and behavior (339). However, in the
unc-64 background, slo-1 causes a small increase in basal
locomotory activity and a dramatic increase in movement
induced by mechanical stimulation. The enhanced activity
of the double mutants suggested that slo-1 mutations
increase neuromuscular neurotransmission. Consistent
with this idea, double mutants were found to have sensitivity to aldicarb similar to that of wild-type animals.
Aldicarb is an inhibitor of acetylcholinesterase that
blocks degradation of acetycholine, causing it to accumulate in neuromuscular junctions. The muscles of wild-type
worms exposed to aldicarb contract tonically resulting in
a hypercontracted paralysis. Mutations in genes such as
unc-64 that inhibit neurosecretion also confer resistance
to aldicarb (e.g., Refs. 232, 283). Thus the suppression of
aldicarb resistance by mutations in slo-1 suggests that the
channel regulates neurotransmitter release.
To examine further the effect of slo-1 on neurotransmission, Wang et al. (339) measured EPG in wild-type and
mutant worms. Three to five IPSPs from M3 glutamatergic
neurons (see sect. IIIF2; Fig. 3D) are visible in EPGs (see
sect. IIC1B) from wild-type worms. IPSPs in EPGs from
unc-64 mutants are greatly reduced in amplitude, indicating that glutamate neurotransmission is reduced. Introduction of slo-1 mutations into unc-64 mutant worms
restores the amplitude of the IPSPs, providing further
evidence that the channel regulates the release of neurotransmitters.
Immunolocalization studies suggested that SLO-1 is
present at synapses and neuromuscular junctions (339).
In addition, Wang et al. (339) observed that rescue of the
double mutant phenotype occurred when wild-type slo-1
was expressed in neurons but not in body wall muscle.
Taken together with the data on aldicarb sensitivity and
pharyngeal electrophysiology, these observations indicate
that the SLO-1 channels function presynaptically to inhibit neurotransmitter release.
To directly assess the effects of slo-1 mutations on
neurotransmitter release, Wang et al. (339) used patch
clamp and the “filleted worm” preparation (see sect. IIC1B,
Fig. 3B) to measure evoked excitatory postsynaptic currents (EPSC) and spontaneous miniature EPSCs
(mEPSC). Quantal content of EPSCs was estimated by
dividing the current integral of an EPSC by the average
current integral of mEPSCs. The slo-1 mutations in wildtype animals increased the duration of EPSCs two- to
threefold and increased quantal content approximately
fourfold. The quantal content of unc-64 mutants was ⬃5%
of that of wild-type worms. In unc-64/slo-1 double mutants, quantal content was increased approximately fourfold due primarily to an increase in EPSC duration. These
ION CHANNEL AND TRANSPORTER BIOLOGY IN C. ELEGANS
tion is a rational and powerful experimental strategy. C.
elegans as well as other nonmammalian model organisms
will undoubtedly continue to provide novel insights into
the integrative physiology of ion channels and transporters as well as numerous other basic biological problems.
These insights will in turn be used as the foundation for
assembling molecular blueprints of more complex organisms including humans.
9.
10.
11.
12.
13.
NOTE ADDED IN PROOF
14.
15.
16.
17.
18.
19.
20.
I am particularly grateful to Drs. Khaled Machaca, Villu
Maricq, Keith Nehrke, and Mike Romero for critically reading
the manuscript and for numerous helpful comments and suggestions. I thank Dr. Ana Estevez for drawing an early version of
Figure 9B.
C. elegans research in my laboratory is supported by National Institute of Diabetes and Digestive and Kidney Diseases
Grants R01-DK-51610, R01-DK-61168, P01-DK-58212, and R21DK-60829.
Address for reprint requests and other correspondence: K.
Strange, Vanderbilt University Medical Center, Anesthesiology
Research Division, T-4202 Medical Center North, Nashville, TN
37232-2520 (E-mail: [email protected]).
21.
22.
23.
24.
25.
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