Ion Homeostasis, Channels, and Transporters:
An Update on Cellular Mechanisms
Experimental Biology 2004: APS Refresher Course
Washington, DC
George R. Dubyak, Ph.D.
Dept of Physiology & Biophysics
Ion Homeostasis, Channels, and Transporters
An Update on Cellular Mechanisms
•
Ion Transport Proteins as Channels versus
Transporters: Not as different as we think
• Interactions of Ion Transport Proteins with Adapter
Proteins: No transporter is an island
• Interactions of Ion Transport Proteins with Local
Lipids: The bilayer as more than a low dielectric
permeability barrier
• Interactions between Ion Transport Proteins and
Modulator Proteins: Cell-specific context explains all
Ion Homeostasis, Channels, and Transporters
An Update on Cellular Mechanisms
Review of Cellular Ion Homeostasis:
Basic Concepts from the “Pre-Genomics” Era
• Ionic Compartments and Electrochemical Driving
Forces
• Ionic Permeability Pathways
• Mechanistic Differences between Ion Channels
and Ion Transporter Proteins (including ATPPowered Pumps)
Basic Concept 1: Compartmentation of Ionic Pools and
Electrochemical Driving Forces
Boron and Boulpaep (2002)
Medical Physiology [Saunders]
Basic Concept 2: Categories of Ion Permeability Pathways
Lodish et al. (2000) Molecular Cell Biology
4th Edition [W.H. Freeman & Co.]
Basic Concept 3: Disparate Mechanisms for Ion Flux
via Channels versus Transporters - The Channel Story
• Minimal energetic
interaction between the
transported ion and the
channel protein
Gadsby (2004) Nature 427: 795-796
• Ionic flux is limited by
opening and closing of a
single major gate
• Gating is regulated by
conformational changes
extrinsic to the
permeability barrier or
pore
Gating is not allosterically coupled
to the movement of ions through
the pore of the open channel
Basic Concept 3: Disparate Mechanisms for Ion Flux via
Channels versus Transporters - The Transporter Story
• There are strong and selective energetic interactions between the
transported ion(s) and the transporter protein
• Ionic flux is limited by the alternating opening and closing of two
gates
• Both gates can be simultaneously closed to produce trapping or
occlusion of the transported ion(s) within the permeability barrier
• Movement of each gate is regulated by conformational changes
intrinsic to the permeability barrier
Basic Concept 3: Disparate Mechanisms for Ion Flux via
Channels versus Transporters - The Transporter Story
Gadsby (2004) Nature 427: 795-796
Gating is allosterically coupled to the movement of ions
through the permeability barrier of the transporter
Basic Concept 3: Disparate Mechanisms for Ion Flux via
Channels versus Transporters
Characterization of channels primarily focuses on:
Ionic selectivity and
permeability of the pore
Boron and Boulpaep (2002)
Medical Physiology [Saunders]
Forces and factors that
move the gates
Lodish et al. (2000) Molecular Cell Biology
4th Edition [W.H. Freeman & Co.]
Basic Concept 4: Disparate Mechanisms for Ion Flux via
Channels versus Transporters
Characterization of transporters primarily focuses on:
Affinity, selectivity,
and stoichiometry of
ion binding
Biochemical or
biophysical reactions
that are allosterically
coupled to the
transport cycle
Gadsby (2004) Nature 427: 795-796
Ion Transport Proteins as Channels versus
Carriers: Not as different as we think
Concept: Both channel-like activity and transporterlike activity can be accommodated within the basic
structures of most transport proteins
Model Example: The induction of channel activity in
the Na+,K+-ATPase pump upon binding of palytoxin,
a marine toxin, that stabilizes both gates of the Na+
pump in the open state
Ion Transport Proteins as Channels versus
Carriers: Not as different as we think
Palytoxin Structure
Hilgemann (2003) PNAS 100: 386-388
Boron and Boulpaep (2002)
Medical Physiology [Saunders]
• Palytoxin is a lethal toxin from a marine coelenterate (Palythoa coral)
• Palytoxin binding to the Na+ pump induces appearance of nonselective cation channel activity
Ion Transport Proteins as Channels versus
Carriers: Not as different as we think
• Model: guinea pig
ventricular myocytes
• Outside-out membrane
patch recording in
symmetric [NaCl]
• Palytoxin (PTX) induces
Na+ channel activity
independent of ATP
Artigas and Gadsby (2003) PNAS 100: 501-505
• However, ATP still acts
as a positive allosteric
regulator
Ion Transport Proteins as Channels versus
Carriers: Not as different as we think
•
K+ acts as a negative allosteric
regulator of PTX-induced Na+
“pump-channels”
Artigas and Gadsby (2003)
PNAS 100: 501-505
•
PTX stabilizes opening of both
gates of the Na+ pump, i.e., no
occluded state
•
Permeability/ conformation of the
non-occluded pore remains
allosterically “sensitive” to ATP
and K+
Boron and Boulpaep (2002) Medical Physiology [Saunders]
Ion Transport Proteins as Channels versus
Carriers: Not as different as we think
Dutzler, Campbell, Cadene,
Chait, and MacKinnon (2002)
Nature 415: 287-294
•
ClC-family proteins comprise a
large family of structurally related
membrane proteins that function
as Cl- channels in eukaryotic cells
•
The resolved crystal of a
prokaryotic member - ClC-ec1
from E. coli - has provided the
structural template BUT…….
A. Accardi and C. Miller (2004)
Nature 427: 803-807
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
Concept: Many channels and transporters physically
associate with adapter proteins that regulate the
subcellular localization of the transport protein
and/or its direct interaction within signal
transduction complexes that include receptors, 2ndmessenger effector enzymes, and protein kinases
Model Example: The role of NHERF-family adapter
proteins in the localization and acute regulation of
Na-Phosphate cotransporters within apical signaling
complexes of renal epithelial cells
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
Ion Transport
Protein
Receptor
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
• PDZ (PSD-95, discs large, ZO1) domains are protein-protein
interaction sites found in a large number of adapter proteins
• Such adapter proteins act to localize channels or transporters within
large signaling complexes at the sub-membrane cytoskeleton
• Examples include the PSD-95 (post-synaptic density) adapter that
co-assembles neurotransmitter-gated ion channels with cytoskeletal
elements, kinases, and small GTPases into signaling complexes at
the neuronal synapses
• NHERFs (Na+/H+ Exchanger Regulatory Factors) comprise another
family of PDZ-containing adapters expressed in many epithelia
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
• Structure of the related
NHERF1 and NHERF2
• ERM domains bind to
cytoskeletal proteins while
PDZ domains bind to various
transport proteins and
signaling proteins
• Recent studies have shown
that NHERFs can also bind the
parathyroid hormone receptor
(PTH-R) and the type 2 NaPhosphate Cotransporter
(NPT2)
PTH-R
NPT2
PLCb1
Shenolikar and Weinman
(2001) Am J Physiol - Renal
280: F389-F395
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
• Phosphate (Pi) reaccumulation
from glomerular filtrate reflects
activity of apical NPT2 protein in
proximal tubule cells
• During phosphate excess, PTH
acts to repress NPT2-mediated Pi
reuptake
• PTH induces rapid endocytosis of
the apical NPT2 pool by signaling
mechanisms that involve both
phospholipase C (PLC) and
adenylyl cyclase (AC) pathways
Boron and Boulpaep (2002)
Medical Physiology [Saunders]
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
OKH +
mut NHERF
•
Opossum Kidney (OK) line cells
exhibit normal PTH-induced
repression of NPT2 -mediated Pi
uptake
•
OKH cells (a subline of OK) exhibit
weak NPT2 response to PTH despite
similar expression of NPT2
•
These effects are correlated with
high NHERF1 levels in OK cells and
low NHERF1 levels in OKH cells
•
Transfection of NHERF1 into OKH
cells restores PTH-induced
repression of NPT2 activity
OKH
OK-wt
OKH +
NHERF
Mahon, Cole, Lederer, and Segre (2003)
Mol Endocrin 17: 2355-2364
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
•
In absence of PTH, NPT2 is
apically expressed in both OKH
cells and OKH transfected with
NHERF1 (OKH-N1)
•
PTH induces rapid NPT2 (aka NaPi-4) internalization in OKH-N1
cells but not OKH cells
Mahon, Cole, Lederer, and Segre (2003)
Mol Endocrin 17: 2355-2364
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
•
Knockout of NHERF1 in mice
induces reduced steady-state levels
of NPT2 in brush-border membranes
from kidney but not reduction in total
kidney NPT2 content
•
Proximal tubule cells from NHERF1 /- mice show increased internal pools
of NPT2 at steady-state
•
NHERF1 -/- mice exhibit exhibit
significant phosphate wasting into
urine despite normal serum Pi levels
•
Most NHERF1 -/- females die within
35 days post birth and show multiple
bone fractures
Shenolikar, Voltz, Minkoff, Wade, and
Weinman (2002) PNAS 99: 11470-11475
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
NHERF association with PTH receptor directs
coupling to distal G proteins and effector enzymes
PTH-R alone
PTH-R
Gs
Adenylyl
Cyclase
PTH-R complexed to NHERF1/2
PTH-R
Gi/q
Phospholipase C
Interactions of Ion Transport Proteins with
Adapter Proteins: No transporter is an island
•
NHERF 1/2 bind to a novel
PDZ-interaction site at the
PTH-R C-terminus
•
Expression of wildtype
PTH-R in absence of
NHERF induces default
coupling to Gs/ AC
•
Coexpression of wildtype
PTH-R with NHERF
redirects coupling to Gi/Gq/
PLC
PTH-R
Gs
Adenylyl
Cyclase
PTH-R /
NHERF
Gi/q
Phospholipase C
Mahon, Donowitz, and
Segre (2002) Nature 417:
858-861
Interactions of Ion Transport Proteins with Local
Lipids: The bilayer as more than a low dielectric
permeability barrier
Concept: The function of many channels and
transporters is directly modulated by the specific
binding of phosphatidylinositol 4,5-bisphosphate
(PIP2); this facilitates rapid modulation of transport
protein activity by highly localized changes in PIP2
synthesis or degradation
Model Example: The hypersensitization of nociceptive
vanillanoid receptor (VR1) channel activity by
inflammatory mediators that activate phospholipase
C (PLC)-dependent PIP2 breakdown
Interactions of Ion Transport Proteins with Local
Lipids: The bilayer as more than a low dielectric
permeability barrier
PIP2 can positively or negatively modulate activity of a wide
range of ion channels and ion transporters
These effects usually reflect direct binding
of PIP2 to specific domains of the
transport proteins with consequent
allosteric modulation
Hilgemann, Feng, and Nasuhoglu (2001)
Science-STKE 111-RE19: 1-8
Interactions of Ion Transport Proteins with Local
Lipids: The bilayer as more than a low dielectric
permeability barrier
Interactions of Ion Transport Proteins with Local
Lipids: The bilayer as more than a low dielectric
permeability barrier
•
Vanillanoid receptors are
non-selective cation
channels of sensory nerve
endings that are gated by
diverse nociceptive stimuli
produced at damaged
tissue
•
The major physiological
stimuli are acidic pH and
increased temperature
•
Sensitivity of VR1 to these
stimuli can be greatly
enhanced by diverse
hydrophobic ligands, e.g.
capsaicins from peppers
Caterina and Julius (2001)
Annu Rev Neurosci 24: 487-517
Interactions of Ion Transport Proteins with Local
Lipids: The bilayer as more than a low dielectric
permeability barrier
•
Model: VR1 channels
heterologously
expressed in HEK293
cells
•
VR1 channels gated by
threshold levels of
primary stimuli (acid or
capsaicin)
•
Activation of native
bradykinin receptors
(PLC-coupled) greatly
potentiates gating by
primary stimuli
Chuang, Prescott, Kong, Shields, Jordl, Basbaum,
Chao, and Julius (2001) Nature 411: 957-962
Interactions of Ion Transport Proteins with Local
Lipids: The bilayer as more than a low dielectric
permeability barrier
O’Neill and Brown (2003) News Physiol Sci 18: 226-231
•
VR1 channels belong to
TRP superfamily
•
VR1 C-terminus contains
sequences conserved in
PIP2-interaction domains
of other PIP2-sensitive
channels, e.g. inward
rectifier K+ channels
Interactions of Ion Transport Proteins with Local
Lipids: The bilayer as more than a low dielectric
permeability barrier
Mutation of this VR1 C-terminal domain increases gating by primary
nociceptive stimuli but reduces potentiation of gating by PLCactivating secondary stimuli
VR1 C-terminal
variants
Prescott and Julius (2003) Science 300: 1284-1288
Interactions among Ion Transport Proteins and Modulator
Proteins: Cell-specific context explains all
Concept: An ion transport protein can exhibit tissuespecific differences in function that reflect its direct
association with modulator proteins that are
expressed in a tissue-specific or stimulus-specific
manner.
Model Example: The role of FXYD-family membrane
proteins in the tissue-specific modulation of Na+,K+ATPase pump activity
Interactions among Ion Transport Proteins and Modulator
Proteins: Cell-specific context explains all
Interactions among Ion Transport Proteins and Modulator
Proteins: Cell-specific context explains all
FXYD proteins as tissue-specific modulators of the Na+,K+-ATPase
Crambert and
Geering (2003)
Science-STKE
166-RE1: 1-9
Interactions among Ion Transport Proteins and Modulator
Proteins: Cell-specific context explains all
•
FXYD proteins
comprise a family of
structurally related type1 membrane proteins
that are expressed in
tissue-specific patterns
•
Includes previously
identified, but poorly
understood proteins,
e.g., phospholemman
(PLM) in heart and
corticosteroid hormoneinduced factor (CHIF) in
kidney
Crambert and Geering (2003
Science-STKE 166-RE1: 1-9
Interactions among Ion Transport Proteins and Modulator
Proteins: Cell-specific context explains all
FXYD proteins as tissue-specific modulators of the Na+,K+-ATPase:
Different effects of different FXYDs
Crambert and Geering (2003
Science-STKE 166-RE1: 1-9
Interactions among Ion Transport Proteins and Modulator
Proteins: Cell-specific context explains all
Treatment of collecting duct
with aldosterone coordinately
increases CHIF and ENaC
expression; CHIF increases Na
affinity of the Na+ pump to
increase transcellular Na flux
while decreasing cytosolic [Na+]
Crambert and Geering (2003
Science-STKE 166-RE1: 1-9
Take-Home Lessons
• Precise homeostasis of the major inorganic cations
(Na+, K+, H+) and anions (Cl-, PO43-, HCO3-) is
fundamental to all cells
• However, cell-specific expression of different
membrane transport proteins and regulatory factors
permits wide variations in the absolute rates of
transmembrane flux of these ions
• These cell-specific differences in ionic flux are
exploited for tissue-specific differences in function
such as solute flow (e.g. transepithelial movements of
metabolites) or information transfer
Take-Home Lessons
• These tissue-specific differences in ionic flux are
regulated at multiple levels:
– via increased/ decreased expression of membrane
transport protein genes
– via changes in the steady-state trafficking of membrane
transport protein to and from the plasma membrane
– via direct post-translational modification (e.g.
phosphorylation) of the membrane transport proteins
– via direct association with tissue-specific adapter or
modulator proteins
– via the local lipid composition of the membrane bilayer
References: Original Research Papers
•
•
•
•
•
•
•
•
Accardi and Miller (2004) Nature 427: 803-807
Artigas and Gadsby (2003) PNAS 100: 501-505
Chuang, Prescott, Kong, Shields, Jordl, Basbaum, Chao, and Julius (2001)
Nature 411: 957-962
Dutzler, Campbell, Cadene, Chait, and MacKinnon (2002) Nature 415: 287294
Mahon, Cole, Lederer, and Segre (2003) Mol Endocrin 17: 2355-2364
Mahon, Donowitz, and Segre (2002) Nature 417: 858-861
Prescott and Julius (2003) Science 300: 1284-1288
Shenolikar, Voltz, Minkoff, Wade, and Weinman (2002) PNAS 99: 1147011475
References: Reviews and Commentaries
• Channel versus Transporter Mechanisms
– Hilgemann (2003) PNAS 100: 386-388
– Gadsby (2004) Nature 427: 795-796
• Adapter/ PDZ Proteins and Channel/ Transporter Regulation
– Shenolikar and Weinman (2001) Am J Physiol - Renal 280: F389-F395
– Noury, Grant, and Borg (2003) Science-STKE 179-RE7: 1-12
• PIP2 and Channel/ Transporter Regulation
– O’Neill and Brown (2003) News Physiol Sci 18: 226-231
– Hilgemann, Feng, and Nasuhoglu (2001) Science-STKE 111-RE19: 1-8
– Caterina and Julius (2001) Annu Rev Neurosci 24: 487-517
• Modulator/ FXYD Proteins and Channel/ Transporter Regulation
– Cornelius and Mahmmoud (2003) News Physiol Sci 18: 119-124
– Crambert and Geering (2003) Science-STKE 166-RE1: 1-9
References: Textbooks
•
Boron and Boulpaep (2002) Medical Physiology [Saunders]
•
Alberts et al. (2001) Molecular Biology of the Cell, 4th Edition [Garland]
•
Lodish et al. (2000) Molecular Cell Biology, 4th Edition [W.H. Freeman &
Co.]