Physiol Rev
82: 893–922, 2002; 10.1152/physrev.00013.2002.
Ryanodine Receptor Calcium Release Channels
MICHAEL FILL AND JULIO A. COPELLO
Department of Physiology, Loyola University Chicago, Maywood, Illinois
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Introduction
History
Structure-Function Overview
Excitation-Contraction Coupling
A. DHPR-RyR interaction
Study of Intracellular Calcium Release
Study of Single Ryanodine Receptor Channels
Permeation Properties
Action of Ryanodine
Overview of Ryanodine Receptor Regulation
A. Cytosolic Ca2⫹
B. Luminal Ca2⫹
C. ATP and Mg2⫹
D. Redox status
E. Cyclic ADP-ribose
F. Phosphorylation/dephosphorylation
Regulation by Closely Associated Proteins
A. Calmodulin
B. Calsequestrin
C. FK-506 binding protein
D. DHPR-loop peptide
Calcium Regulation Paradox
Steady-State Calcium Dependence
Calcium Activation Kinetics
A. Rate of Ca2⫹ activation
B. Rate of Ca2⫹ deactivation
Feedback Calcium Regulation
Potential Negative Control Mechanisms
A. Ca2⫹-dependent inactivation
B. Stochastic attrition
C. Adaptation
D. Activation-dependent or “fateful” inactivation
E. Depletion/luminal Ca2⫹ effects
F. Allosteric or coupled gating
Adaptation Debate
A. DM-nitrophen Ca2⫹ overshoot
B. Ca2⫹ overshoot concern
C. Differing interpretations
Modal Gating
Concluding Remarks
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Fill, Michael, and Julio A. Copello. Ryanodine Receptor Calcium Release Channels. Physiol Rev 82: 893–922,
2002; 10.1152.physrev.00013.2002.—The ryanodine receptors (RyRs) are a family of Ca2⫹ release channels found on
intracellular Ca2⫹ storage/release organelles. The RyR channels are ubiquitously expressed in many types of cells
and participate in a variety of important Ca2⫹ signaling phenomena (neurotransmission, secretion, etc.). In striated
muscle, the RyR channels represent the primary pathway for Ca2⫹ release during the excitation-contraction coupling
process. In general, the signals that activate the RyR channels are known (e.g., sarcolemmal Ca2⫹ influx or
depolarization), but the specific mechanisms involved are still being debated. The signals that modulate and/or turn
off the RyR channels remain ambiguous and the mechanisms involved unclear. Over the last decade, studies of
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0031-9333/02 $15.00 Copyright © 2002 the American Physiological Society
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MICHAEL FILL AND JULIO A. COPELLO
RyR-mediated Ca2⫹ release have taken many forms and have steadily advanced our knowledge. This robust field,
however, is not without controversial ideas and contradictory results. Controversies surrounding the complex Ca2⫹
regulation of single RyR channels receive particular attention here. In addition, a large body of information is
synthesized into a focused perspective of single RyR channel function. The present status of the single RyR channel
field and its likely future directions are also discussed.
I. INTRODUCTION
Physiol Rev • VOL
II. HISTORY
The significance of SR Ca2⫹ release during skeletal
muscle contraction was recognized many decades ago
(for review, see Refs. 38, 42, 260). Electrophysiological
experiments and 45Ca autoradiography studies provided
evidence in intact muscle that Ca2⫹ is released from the
terminal cisternae of the SR in response to depolarization
of surface membrane invaginations called transverse tubules (t tubules) (reviewed in Ref. 260). A number of
studies followed, and a putative mechanism was proposed (43, 251–255, 265–267, 296), namely, that there is a
physical coupling between an integral t-tubule protein
and the SR Ca2⫹ release channel. The idea was that
voltage-dependent movements of charged particles in the
integral t-tubule protein activate the SR Ca2⫹ release
channel through this physical coupling. This mechanism
is generally referred to as depolarization-induced Ca2⫹
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Calcium is a common second messenger (38, 42, 123)
that regulates many processes in cells (e.g., contraction,
secretion, synaptic transmission, fertilization, nuclear
pore regulation, transcription). Although cells are typically bathed in solutions containing a relatively high Ca2⫹
concentration (i.e., in the millimolar range), the cytoplasm of most cells contains much lower resting Ca2⫹
concentrations (i.e., in the 100 nM range). Thus Ca2⫹
entry across the surface membrane can substantially elevate cytosolic Ca2⫹ levels providing the Ca2⫹ trigger signals for a large number of physiological processes. The
surface membrane, however, is not the only source of
triggering Ca2⫹ signals. Most cells have developed an
additional pathway to generate localized and fast trigger
Ca2⫹ signals deep inside the cell. This other pathway
involves specialized intracellular Ca2⫹ storage/release organelles.
Cells have many different types of Ca2⫹ transporters
(channels, exchangers, pumps) that modulate cytosolic
Ca2⫹ levels. The rich array of Ca2⫹ transport proteins in
the surface and intracellular membranes provides numerous potential Ca2⫹ signaling pathways that can respond to
many different types of stimuli. Specificity of any one
Ca2⫹ signal pathway to a particular process is often made
possible by assembling the particular signaling proteins
into a local complex. Localizing the Ca2⫹ signaling effectively allows Ca2⫹ to be effectively directed to a particular
physiological function. Certain vascular smooth muscles
are a good example of this. In these smooth muscles, large
global intracellular Ca2⫹ signals activate the contractile
machinery causing the cell to contract. However, small
very localized Ca2⫹ signals (i.e., Ca2⫹ sparks) that arise
from intracellular Ca2⫹ stores activate certain K⫹ channels in the surface membrane promoting relaxation of the
cell (219).
The primary intracellular Ca2⫹ storage/release organelle in most cells is the endoplasmic reticulum (ER).
In striated muscles, it is the sarcoplasmic reticulum (SR).
The ER and SR contain specialized Ca2⫹ release channels.
There are two families of Ca2⫹ release channels: the
ryanodine receptors (RyRs) and inositol trisphosphate
receptors (IP3Rs). Each family of Ca2⫹ release channels
appears to contain three different isoforms (300, 309).
However, there may be a fourth type of RyR channel in
fish (i.e., RyR-slow; Refs. 90, 210). The Ca2⫹ release channels are large oligomeric structures formed by association
of either four RyR proteins or four IP3R proteins. The RyR
and IP3R proteins share significant homology, and this
homology is most marked in the sequences that may form
the channel’s pore (204, 271, 299, 315a, 367).
Calcium regulates all the RyR and IP3R channels.
However, each Ca2⫹ release channel has its own distinguishing functional attributes. The IP3R channels require
the presence of inositol 1,4,5-trisphosphate. The activity
of one type of RyR channel is coupled to a “voltage
sensor” in the plasma membrane. The Ca2⫹-induced Ca2⫹
release process governs the activity of the other types of
RyR channel. The distinguishing functional attributes of
each RyR or IP3R channels likely underlie the spatiotemporal complexity of intracellular Ca2⫹ signaling in cells.
During the last 10 years, there have been numerous
reviews on several aspects of regulation of RyR-mediated
Ca2⫹ signaling and/or related topics (e.g., Refs. 18, 43, 60,
69, 76, 84, 87, 92, 112, 179, 195, 199, 202, 225, 245, 252, 253,
255, 260, 265, 281, 287, 300, 301, 307, 315a, 335, 346, 354).
Here, we focus on the RyR Ca2⫹ release channels found in
striated muscle. The controversial and complex Ca2⫹ regulation of single RyR channels is given particular attention. An attempt has been made to cover this large rapidly
growing and dynamic body of information. Regrettably, it
is impossible to describe all of the quality studies that
characterize this field. It is hoped that the single-channel
perspective of this review will be useful to readers seeking to understand the complex dynamics of single RyR
channel regulation
RYANODINE RECEPTOR Ca2⫹ REGULATION
Physiol Rev • VOL
muscle, ryanodine promotes Ca2⫹ leak from intracellular
stores because it locks the RyR channels in the long-lived
subconduction state. This “leaked” Ca2⫹ is rapidly removed from the cell by the relatively strong surface membrane Ca2⫹ extrusion mechanisms present in cardiac
muscle (17, 20). Intracellular Ca2⫹ stores eventually deplete, yielding the observed flaccid cardiac paralysis. In
contrast, skeletal muscle lacks the strong surface membrane Ca2⫹ extrusion mechanisms present in cardiac
muscle. The ryanodine-induced Ca2⫹ leak from intracellular stores causes Ca2⫹ to accumulate in the cytoplasm.
The result is abnormally high cytoplasmic Ca2⫹ levels that
induce sustained contraction (i.e., the rigid skeletal muscle paralysis).
III. STRUCTURE-FUNCTION OVERVIEW
Molecular cloning studies have defined three different RyR isoforms in fish, amphibian, birds, and mammals
(6, 60, 205, 215, 225, 245, 301, 307, 315a). Although the RyR
proteins share significant homology with the IP3R receptors, they have little homology with the more widely
studied voltage-dependent Ca2⫹ channels found in the
surface membrane (204, 315a, 321). In mammals, the three
RyR isoforms (RyR1, RyR2, and RyR3) are encoded by
three different genes on different chromosomes (186, 205,
318, 315a, 369). The RyR proteins are in a variety of
tissues, but the highest densities are in striated muscles
(5, 6, 148, 205, 212, 225, 227, 231, 233, 299, 308). There
may also be structurally, and perhaps functionally, distinct splice variants of these channels (102, 221, 370).
However, the functional significance and distribution
among fiber types of RyR splice variants are currently
unknown.
In mammalian striated muscles, the expression of the
different RyR protein isoforms is tissue specific. The predominant RyR isoform in skeletal muscle is the RyR1
protein, commonly referred to as the skeletal RyR isoform
(60, 195, 225, 245, 299). The RyR2 protein is the most
abundant isoform in cardiac muscle, and the RyR2 protein
is commonly referred to as the cardiac RyR isoform. The
RyR3 protein is also found in mammalian striated muscles, but at relatively low levels (98, 308, 325). For example, the RyR3 protein represents ⬍5% of the overall RyR
population in diaphragm (135, 213).
What is the physiological role of the RyR3 channel in
striated muscle or elsewhere? Mice missing the RyR1 and
RyR2 gene products (i.e., RyR1 or RyR2 knock-outs) die
early during embryonic development (317, 320). In contrast, mice missing the RyR3 gene product (i.e., RyR3
knock-out) lead relatively normal lives and have quite
normal striated muscles (55, 317). Interestingly, the CICR
process in neonate skeletal muscle fibers from RyR3knockout mice appears to be impaired (357). The impli-
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release (DICR). Parallel work in other experimental preparations (i.e., SR vesicles and skinned muscle fibers) confirmed the existence and importance of the DICR mechanism in skeletal muscle (38, 94, 121, 127, 138, 150, 151,
183, 260, 296, 321).
In other early works, it was realized that a small
increase in cytosolic Ca2⫹ concentration could induce
massive intracellular SR Ca2⫹ release events in both skeletal and cardiac muscle (75, 89). This phenomenon is
often called Ca2⫹-induced Ca2⫹ release (CICR). Later,
meticulous studies by Fabiato (80, 81) established that the
CICR process was sensitive to both the speed and amplitude of the applied Ca2⫹ trigger. These studies and others
helped reveal the complexity of the regulatory mechanisms that control SR Ca2⫹ release.
A major advancement was the identification of the SR
Ca2⫹ release channel using an alkaloid called ryanodine.
Ryanodine is found naturally in the stem and roots of the
plant Ryania speciosa. The ryanodine alkaloid was first
isolated as a potential insecticide (73, 133), and its potent
paralytic action on skeletal and cardiac muscles was immediately evident (73, 82, 93, 218, 311, 312). However, its
action was difficult to understand. Specifically, it was
difficult to understand how ryanodine could induce rigid
paralysis in skeletal muscle but flaccid paralysis in cardiac muscle.
The ryanodine alkaloid was shown to inhibit SR Ca2⫹
release by binding with high affinity to a protein present in
the SR membrane (41, 80, 88, 93, 309 –312, 345, 347). The
ryanodine binding protein was purified (39, 88, 124, 147,
240, 241) and existed in a tetrameric complex (149) whose
shape was subsequently visualized using electron microscopy (131, 147). Interestingly, the size and shape of the
ryanodine binding protein complex was similar to that of
the “feet” structures, which appear to physically link the t
tubule and SR membranes (83, 94). Incorporation of the
ryanodine binding protein complex into artificial planar
lipid bilayers revealed that this structure was an ion channel (124, 130, 147, 294). The RyR channel is a poorly
selective Ca2⫹ channel (selectivity Ca2⫹/K⫹ ⬃6) with very
high conductance (⬃700 pS when K⫹ is charge carrier
and ⬃100 pS when Ca2⫹ is charge carrier). The channel is
regulated by Ca2⫹, Mg2⫹, ATP, and caffeine. Interestingly,
application of the ryanodine alkaloid locks the channel in
a slow-gating subconductance state (124, 147, 294, 337).
Openings of the ryanodine-bound channel are long-lived
and have a smaller unit current (⬃1/3 or ⬃1/2 of control
amplitude). This action of ryanodine on single RyR channel function is quite distinctive and is now often used to
functionally identify the channel.
These early single-channel studies unambiguously
identified the RyR channel as the SR Ca2⫹ release channel
in striated muscle (84, 87). It also became evident how the
ryanodine alkaloid can induce rigid paralysis in skeletal
muscle but flaccid paralysis in cardiac muscle. In cardiac
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MICHAEL FILL AND JULIO A. COPELLO
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tive TMRs generates channels with altered ion selectivity
(23, 103, 367). Thus there is reasonable certainty that this
region of the protein contains the determinants that form
the Ca2⫹-selective pore of the RyR channel.
Analysis of the RyR’s primary amino acid sequence
also has revealed several consensus ligand binding (e.g.,
ATP, Ca2⫹, caffeine, and calmodulin) and phosphorylation motifs. Experimental verification of these sequences
as potential regulatory sites (e.g., Refs. 23, 45, 47) is
ongoing. For example, substitution of alanine-3882 by a
glutamate (E3882A) in the RyR3 protein results in RyR3
channels with altered Ca2⫹ sensitivity (45). This implies
that this region of the protein may contain the “Ca2⫹
sensor” of the channel. A gross deletion of amino acids
183– 4006 in the RyR1 protein results in channels that lack
caffeine sensitivity and do not inactivate at high Ca2⫹
concentrations (23). Exchange of amino acids 4187– 4628
of RyR1 for the corresponding cardiac RyR2 sequence
results in channels with increased caffeine sensitivity and
altered Ca2⫹ sensitivity (66, 67).
Two skeletal muscle diseases, malignant hyperthermia (MH) and central core disease (CCD), have also
provided useful insights into the RyR1 structure-function
relationship (180, 366). These diseases are marked by
pathologies like hypercarbia, rhabdomyolisis, and generalized muscle contraction. A defect in the RyR1 channel
function was suspected to be involved in these pathologies (85, 277, 338). It is now known that specific point
mutations in the RyR1 protein are associated with the MH
and CCD diseases (180, 243, 277, 366). Recent reports
describe more than 20 different point mutations in the
human RyR1 isoform that are associated with the MH
and/or CCD syndromes (140, 193). The majority of the
mutations are clustered in two regions of RyR1 sequence,
residues 35– 614 (near the NH2 terminal) and 2163–2458 (a
central location). These data are important because these
mutations may reveal important points in the RyR1 structure that govern its function.
It is clear that the exploration of the RyR structurefunction relationship by mutagenesis is still in its infancy.
This is in stark contrast to the molecular study of other
types of ion channels. There are several reasons why we
have not progressed further or faster in this area. First,
the RyR channels are expressed in intracellular organelles
(ER or SR), and thus single RyR channel function cannot
be directly and easily accessed. For surface membrane
channels, function is often efficiently assessed by the
classical patch-clamp method on a few tagged expressing
cells. For the RyR case, function is generally defined in
vitro after the channels have been biochemically isolated
from millions of expressing cells. Second, the RyR is a
huge protein (⬃1/10 the size of a ribosome). This large
size complicates its genetic manipulation as well as the
selection of sites to mutate. Third, recent studies suggest
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cations of this observation are not yet clear. In any event,
it appears that the physiological role of the RyR3 channel
in mammals, unlike that of the RyR1 and RyR2 channels,
can be readily compensated for both during development
and in the adult.
The RyR nomenclature and distribution are somewhat different in nonmammalian striated muscle (35, 148,
212, 232, 307). Nonmammalian skeletal muscle (e.g., frog,
fish, and chicken) contains nearly equal amounts of two
different RyR proteins, named the ␣-RyR and ␤-RyR.
These isoforms are homologs of mammalian RyR1 and
RyR3 forms, respectively (5, 6, 148, 212, 223, 227, 233, 308,
307). It is known that embryonic chicken skeletal muscle
cells fail to develop normal excitation-contraction coupling in the absence of ␣-RyR (132), the RyR1 analog.
Although there have been no ␤-RyR knock-out studies, all
indications are that the ␤-RyR must play some fundamentally important role in the operation of nonmammalian
skeletal muscle (307). One likely possibility is that the
␤-RyR isoform participates in the CICR process, effectively amplifying the initial DICR mediated by the ␣-RyR
isoform.
The RyR proteins are also expressed in smooth muscle and many nonmuscle tissues (e.g., neurons). These
other tissues generally contain multiple RyR isoforms.
For example, expression of all three RyR isoforms has
been reported in aortic smooth muscle (162, 186), cerebrum (101, 115, 220), and cerebellum (101, 115, 187).
These tissues also express multiple IP3R isoforms, and
thus one cell may contain many types of RyR and IP3R
channels. The significance of this remarkable redundancy
in intracellular Ca2⫹ release channel expression in nonmuscle systems is not clearly understood. One might
speculate that the rich morphological diversity implies
equally rich functional heterogeneity and that the multiple
functionally distinct Ca2⫹ release channels may govern
different Ca2⫹ signaling tasks. Consequently, Ca2⫹ release
channel diversity may represent one factor that allows
cells to simultaneously carry out the myriad of Ca2⫹
signaling tasks they need to do to survive. Even striated
muscles, which are highly specialized for the Ca2⫹ signaling that governs contraction, must carry out other types
of Ca2⫹ signaling tasks to survive. Perhaps the RyR3
channel or the low density of IP3Rs expressed in striated
muscles (239, 276) carry out this “other” Ca2⫹ signaling.
At the amino acid level, the three mammalian RyR
isoforms share ⬃70% identity (115, 215, 318, 315a). Analysis of the primary amino acid sequences suggests that
the membrane-spanning domains of the RyR are clustered
near its COOH terminus (271, 299, 315a, 367). Truncation
mutants, containing only these putative transmembrane
regions (TMRs), are sufficient to form Ca2⫹-selective
channels when incorporated into planar lipid bilayers (23,
24). Mutation of certain regions in and around the puta-
RYANODINE RECEPTOR Ca2⫹ REGULATION
Physiol Rev • VOL
ecules (⬃9 Å) occupy the single-file region of the RyR2
channel pore (337). Thus the RyR channels appear to have
a relatively short permeation pathway.
IV. EXCITATION-CONTRACTION COUPLING
A role in striated muscle excitation-contraction (E-C)
coupling is probably the RyR channel’s most notable
claim to fame. There is clear evidence that the RyR channels interact with voltage-dependent Ca2⫹ channels (i.e.,
dyhydropyridine receptors; DHPRs) in the nearby t-tubule
membrane (246, 247, 321, 322). This functional interaction
between the DHPR and RyR is commonly referred to as
E-C coupling (Figs. 1 and 2). Depolarization of the t-tubule
membrane (i.e., excitation) induces conformational
changes in DHPR that ultimately lead to activation of RyR
channel in the SR membrane. The activation of RyR channels leads to massive Ca2⫹ release from the SR, which in
turn initiates contraction. The bulk of information addressing RyR channel regulation has been obtained from
studies focused on the process of E-C coupling in striated
muscle.
More than a century ago, Ringer demonstrated that
the cardiac E-C coupling process required the presence of
extracellular Ca2⫹ (reviewed in Ref. 38). In cardiac muscle, the DHPR receptor (an L-type Ca2⫹ channel) carries a
small Ca2⫹ influx that activates the RyR2 channel (16, 208,
250, 270, 272). A similar process governs the RyR channels in some invertebrate skeletal muscles (177, 260).
However, this is not the case in most vertebrate skeletal
muscle, where extracellular Ca2⫹ is not required for E-C
coupling (9, 38, 91, 129, 177, 260). The primary role of the
DHPR in vertebrate skeletal muscles is acting as a voltage
sensor that directly (perhaps physically) modulates the
activation gate of nearby RyR1 channels.
A. DHPR-RyR Interaction
In skeletal muscle (Fig. 1), the DHPR and RyR1 are
thought to communicate via some sort of physical protein-protein linkage (39, 246, 247, 251, 253, 265). The
membranes of the t tubule and SR are juxtaposed and
separated by a small 10-nm gap. The cytosolic domain of
the RyR1 channel spans this narrow gap (28, 29, 94 –97).
Electron microscopy (EM) studies show that the skeletal
DHPR in the t tubules are arranged in clusters of four
(tetrads). These tetrads are organized into distinct arrays.
The RyR1 channels in the SR membrane are arranged in a
corresponding fashion (28, 29, 94 –97, 247). The arrays of
DHPR and RyR align in certain fast-twitch skeletal muscle
such that every other RyR1 channel is associated with a
DHPR tetrad (28, 29, 247).
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that RyR1 channel function may involve inter-RyR domain
interactions (128, 279, 355), and thus some functional
attributes may be controlled by multiple and very complex structural determinants. Despite these and other
technical challenges, future mutagenesis studies promise
to provide some very interesting insights into RyR structure-function in the not too distant future.
Usually, ion channel structure-function studies are
interpreted in the absence of information concerning the
channel’s detailed three-dimensional (3-D) structure.
There are some exceptions where channels or channel
segments have been crystallized. However, the RyR channel is not one of these exceptions. Intriguingly, some
gross details of the 3-D RyR structure (i.e., general shape)
have been predicted using cryoelectron microscopy and
3-D reconstruction techniques. The RyR1 channel has
fourfold symmetry, presumably reflecting its formation by
four RyR protein monomers (249, 270, 342). The RyR
channel has two distinct domains (249, 270, 342). One is a
large cytoplasmic assembly (⬃29 ⫻ 29 ⫻ 12 nm), consisting of loosely packed protein densities. The other is a
small transmembrane assembly that protrudes (⬃7 nm)
from the center of the cytoplasmic assembly (270, 342).
This transmembrane assembly appears to have a central
hole that can be occluded by a pluglike mass. This hole
and plug may correspond to the Ca2⫹-selective pore and
its gate (230). In general, the shapes of the RyR1 and RyR2
channels are quite similar (275). There are, however, specific points of structural heterogeneity. These points may
contribute to the isoform-specific functional attributes of
the RyR channels. Cryoelectron microscopy studies have
also revealed the sites where certain peptides (calmodulin, FK binding proteins, and imperatoxin) interact with
the RyR channels (263, 343). Recently, antibodies have
been used to map the location, on the 3-D structure, of a
specific domain (amino acid residues 4425– 4621) that
may be implicated in the Ca2⫹-dependent regulation of
the RyR channel (15).
Certain structural details of the RyR channel have
also been obtained using certain biophysical approaches.
For example, the width of the RyR2 pore has been estimated from molecular sieving experiments (332). In these
studies, it was found that only cations with a predicted
circular radius smaller than 3.5 Å could permeate through
the channel. In another study, streaming potential experiments indicated that the RyR2 channel contains a narrow
region in which H2O (radius 1.5 Å) and Cs⫹ (radius 2 Å)
must pass in a single-file fashion (337). Combined, these
results suggest that the RyR2 channel pore is ⬃3 Å wide.
Electrical distance measurements, using trimethylammonium derivatives of varying length, indicated that the
physical length of the voltage drop along the RyR2 channel pore is ⬃10.4 Å (331). This is consistent with steaming
potential measurements that suggest that three H2O mol-
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MICHAEL FILL AND JULIO A. COPELLO
It is thought that the skeletal DHPR physically contacts the large cytoplasmic domain of the juxtaposed
RyR1 channel to form a linkage through which t-tubule
voltage-sensing information is transduced (246, 251–255,
265–267). A distinctive feature of this transduction mechanism is its speed. Signal transmission between the skeletal DHPR and RyR1 channel must occur during the very
brief (⬃2 ms) skeletal muscle action potential. It may be
FIG. 2. Environment of the RyR2
channel in cardiac muscle. The t tubule
contains DHPRs and calcium extrusion
mechanisms like the Na⫹/Ca2⫹ exchanger (Na-CaX). A t-tubule membrane
voltage change activates the DHPR, and
the resulting calcium entry triggers underlying RyR2 channels to open. The
open RyR2 channel releases the calcium,
stored inside the SR, into the cytoplasm.
CSQ, a low-affinity high-capacity calcium
buffer, buffers calcium near the RyR2
channels inside the SR. The SERCA uses
the energy stored in the ATP to pump
calcium back into the SR. The Na-CaX
uses the energy stored in the sodium gradient to move calcium out of the cell.
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FIG. 1. Environment of the ryanodine
receptor channel in skeletal muscle. The
t tubule contains tetrads of dihydropyridine receptors (DHPRs). Every other
RyR1 channel is associated with a DHPR
tetrad. A t-tubule membrane voltage
change (⌬VM) activates the DHPR. Calcium entry thorough the DHPR is not
important to RyR1 channel activation in
skeletal muscle. Instead, the t-tubule ⌬VM
signal is thought to be transmitted to the
RyR1 channel by a direct physical DHPRRyR1 linkage. A specific cytosolic loop of
the DHPR is thought to be involved in
this physical communication. The t-tubule ⌬VM signal triggers the RyR1 channel to open and release the calcium
stored inside the sarcoplasmic reticulum
(SR). Calsequestrin (CSQ), a low-affinity
high-capacity calcium buffer, is found inside the SR lumen near the RyR1 channels. The SR Ca2⫹-ATPase (SERCA) uses
the energy stored in the ATP to pump
calcium back into the SR.
RYANODINE RECEPTOR Ca2⫹ REGULATION
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V. STUDY OF INTRACELLULAR
CALCIUM RELEASE
The first methods used to study Ca2⫹ transport
across cell membranes monitored radioisotope (45Ca) influx/efflux between two defined compartments (e.g., bath
and cytosol; Ref. 141). From these initial studies, it became clear that Ca2⫹ was sequestered into intracellular
organelles and that the cytosol represented an intermediary compartment with variable Ca2⫹ buffer properties.
The next generation of studies examined Ca2⫹ influx/
efflux across the SR of mechanically or chemically
skinned muscle fibers. Here, the barrier of the plasma
membrane was eliminated and the cytosolic solution
could be controlled. The importance of ATP-mediated
Ca2⫹ uptake by the SR and some of the attributes of CICR
and even DICR were defined (80, 81, 150, 153, 151). Almost concurrently, methods for generating subcellular
fractionation of functional SR vesicles were developing.
Studies of Ca2⫹ loading and release from suspensions of
SR vesicles provided improved temporal resolution and
further revealed the intricacies of the SR Ca2⫹ release
process (121, 127, 138, 196, 198, 199, 234 –236).
It was noticed by several investigators that the binding of the ryanodine alkaloid appeared to depend on the
activity level of the SR Ca2⫹ release channel (e.g., Refs.
53, 122). Experimental conditions that promoted SR Ca2⫹
release increased ryanodine binding. Experimental conditions that inhibited SR Ca2⫹ release decreased ryanodine
binding. Subsequently, “nonequilibrium” ryanodine binding became a commonly used tool to assess RyR channel
activity (discussed in detail in Ref. 225). Thus a number of
methods have been developed to study the SR Ca2⫹ release phenomenon in populations of RyR channels.
The development of the patch-clamp technique revolutionized the study of ion channels, including the study
of the RyR channel. In this classic method, a small glass
pipette is placed on the surface of a cell to physically
isolate a small patch of membrane in which the function
of an individual ion channel can be measured. This method
has been widely applied to define the function of surface
membrane ion channels. The patch-clamp technique,
however, cannot be easily applied to study ion channels in
intracellular organelles. Instead, microsomes isolated
from cellular homogenates are fused into artificial planar
lipid bilayers (206). The same instrumentation used to
record across the membrane patch is then used to record
across the artificial bilayer. Thus studies of single RyR
channel behavior became possible (detailed in sect. VI).
The recent development of laser scanning confocal
microscopy opened yet another chapter in the study of
intracellular Ca2⫹ release. Confocal imaging has made it
possible to visualize very localized intracellular Ca2⫹ release events (Ca2⫹ sparks; Refs. 50, 336). These local
events may arise from the opening of an individual chan-
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this “need for speed” that has converted the physiological
role of the DHPR from Ca2⫹ channel to voltage sensor in
mammalian skeletal muscle.
In cardiac muscle (Fig. 2), there is about 1 DHPR for
every 5–10 RyR2 channels (29, 247, 313), and the DHPR
and RyR2 channels are not aligned in such a highly ordered fashion (29, 95, 247, 313). During the long cardiac
action potential (⬃100 ms), the DHPR Ca2⫹ channel has
ample time to open and mediate a substantial Ca2⫹ influx.
This Ca2⫹ influx is ultimately the signal that activates the
underlying RyR2 channels via the CICR process (18, 40,
272). The involvement of a diffusible second messenger
(i.e., Ca2⫹) makes the DHPR-RyR2 signaling considerably
slower than the DHPR-RYR1 signaling. This lack of speed,
however, may provide a greater opportunity for regulating
DHPR-RyR2 signaling. In fact, pharmacological regulation
of DHPR-RyR2 signaling is fundamental to normal cardiac
function (18).
The expression of mutant DHPRs in mouse skeletal
muscle myotubes that lack the endogenous DHPR has
provided insights into which regions of the DHPR are
involved in DHPR-RyR1 interaction (1, 2, 106, 321). A
region within the cytosolic II-III loop of the DHPR’s ␣-subunit (residues 720 –765) appears to be critical. Furthermore, studies with myotubes lacking endogenous RyR1
channels showed that RyR2 or RyR3 could not substitute
for RyR1 in the DHPR-RyR interaction (216, 320, 356). The
expression of chimeric RyR channels (RyR1/RyR2) indicates that regions R9 (2659 –3720) and R10 (1635–2636) of
RyR1 contain sequences involved in the DHPR-RyR1 interaction (217, 246). Thus specific regions of the DHPR
and RyR1 channels have been implicated in the DHPRRyR1 interaction. How these regions mediate the required
signal transduction remains to be determined.
The DHPR is not the only regulator of RyR1 channel
function. Like the RyR2 channel, the RyR1 channel (in the
absence of the DHPR) can be activated by cytosolic Ca2⫹
signals. This is important because more than half of the
RyR1 channels are not coupled to DHPRs (7, 183, 197,
200). These “uncoupled” RyR1 channels are thought to be
available to participate in the CICR process. It is generally
believed that CICR acts to amplify the DICR signal (i.e.,
that generated by the DHPR-RyR1 interaction). Interestingly, the presence of “coupled” and uncoupled RyR1
channels implies that RyR1 function is inherently heterogeneous “in vivo.” The amount of uncoupled RyR1 channels is not the same in all skeletal muscles. Slow-twitch
skeletal muscles may have three or more uncoupled RyR1
channels for each DHPR-linked RyR1 channel (7, 183, 197,
200). This may mean that these muscles have a greater
CICR component. This may be related to the substantially
slower rate of E-C coupling in slow-twitch muscle (104,
223, 227, 260, 370).
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MICHAEL FILL AND JULIO A. COPELLO
nel or more likely the concerted opening of a small number of RyR channels.
VI. STUDY OF SINGLE RYANODINE
RECEPTOR CHANNELS
Physiol Rev • VOL
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Defining the characteristics of single RyR channels is
fundamental to understand the origin of many intracellular Ca2⫹ signaling phenomena. The amplitude of single
RyR channel current reveals the number of Ca2⫹ passing
through the channel per second. The open probability
(Po) of single RyR channels allows us to predict how
certain agonists and antagonists regulate its activity.
Dwell-time analysis of single RyR channel activity reveals
the durations of the open and closed events. Kinetic studies of single RyR channel activity reveal how fast it can
respond to a ligand stimulus (i.e., time constants and first
latencies) as well as its specific patterns of opening (i.e.,
modal gating or bursting behavior). Additionally, single
RyR channel studies can reveal the extent of homogeneity
or heterogeneity of individual channel function in the
overall RyR channel population (56, 60, 178, 238). These
types of information are generally not available when
Ca2⫹ signaling phenomena are studied in a population of
RyR channels. Thus single RyR channel studies provide
detailed information about the origin of intracellular Ca2⫹
signaling phenomena that simply cannot be obtained by
other means.
The study of single RyR channel behavior requires
the incorporation of native SR vesicles (enriched in RyR
channel) or purified RyR channel proteins into an artificial planar lipid bilayer. The artificial planar bilayer is
generally formed across a small aperture that separates
two solutions. A patch-clamp amplifier is used to monitor
electrical signals that arise from the bilayer. Single RyR
channel activity is evident as a sudden appearance of
single-channel activity in the bilayer. The sudden appearance of channel activity marks the moment a channel
protein has been incorporated into the bilayer. This methodology is now widely used and has been described in
detail elsewhere (206).
The environment in which the single RyR channel
operates in the cell is complex (163). The resting cytosolic
free Ca2⫹ concentration is near 100 nM, and the free Ca2⫹
concentration inside the SR is thought to be near 1 mM
(196, 274). The cytosol contains a complex mixture of
salts (⬃140 mM K⫹, ⬃1 mM Mg2⫹, 5–10 mM ATP, and
various other nucleotides; Refs. 234, 371). The cell also
contains several proteins that are known to interact with
the RyR channel (e.g., DHPR, calmodulin, FK binding
proteins, triadin, kinases-phosphatases, calsequestrin, annexin, etc.). Reconstructing or mimicking, at least in part,
this complex situation in vitro (i.e., during a planar bilayer
experiment) still needs to be done. Nearly all the single
RyR channel studies have been done under very simple
experimental conditions that do not reflect the channel’s
physiological reality (e.g., Refs. 54, 56, 116, 145, 147, 291).
This fact should always be taken into consideration when
interpreting single RyR channel data.
How well does single RyR channel function in the
bilayer reproduce the single RyR channel behavior in the
cell? The correlation between single RyR channel function in bilayers and SR Ca2⫹ release phenomena observed
in cells is surprisingly strong. For example, the primary
regulatory features of SR Ca2⫹ release in cells (e.g., its
Ca2⫹, Mg2⫹, and ATP sensitivity) are clearly evident at the
single RyR channel level in bilayers. The actions of many
secondary pharmacological regulatory agents like caffeine, ruthenium red, and ryanodine in the cell are mimicked at the single RyR channel level in bilayers. The fast
gating and high conductance of single RyR channels in
bilayers is consistent with the speed and large amplitude
of intracellular Ca2⫹ release phenomena in cells (173).
There is growing evidence that many regulatory factors
and closely bound regulatory proteins are actually retained when single RyR channels are isolated in native
membranes (e.g., Refs. 30, 189, 287, 300, 346, 348, 365).
Thus defining single RyR channel behavior in bilayers is
likely to provide insights into the origins of RyR-mediated
Ca2⫹ signaling in cells.
The RyR channel is usually isolated by differential
centrifugation of muscle homogenates. The RyR channel
is found in heavy microsomal fractions. The RyR-containing vesicles (microsomes) can then be fused into planar
lipid bilayers (36, 54, 56, 258, 291, 298). Alternatively, the
RyR-containing vesicles can be detergent-solubilized
(CHAPS plus phospholipids mixtures) and the resulting
“purified” RyR channels can then be directly fused in a
lipid bilayer (124, 135, 238) or reconstituted into liposomes (145, 147). These proteoliposomes can then fuse
into planar lipid bilayers. Single RyR channel function has
been examined using all these approaches. An advantage
of using the SR vesicle approach is that the RyR channel
is present in its native membrane, increasing the likelihood that closely associated regulatory proteins/factors
may still be present (e.g., Refs. 189, 327). A disadvantage
of using the SR vesicle approach is that the vesicles may
contain other types of ion channels, specifically K⫹ and/or
Cl⫺ channels. The presence of other channels complicates things considerably. Potential interference from
other channels is minimal if Ca2⫹ (or Ba2⫹) is used as the
charge carrier (with no other permeant ions present).
Many single RyR channel studies, however, chose to use
a monovalent charge carrier to boost signal-to-noise characteristics. In this case, carefully selected ionic substitutions are usually made to eliminate potential contamination from other channel types.
RYANODINE RECEPTOR Ca2⫹ REGULATION
VII. PERMEATION PROPERTIES
The ability of the RyR channel to mobilize intracellular Ca2⫹ depends on both its permeation and gating
properties. Its permeation properties include its unitary
conductance and ion selectivity. Its gating properties include its Po and the duration of individual open/closed
events. A single RyR channel with optimal permeation
properties would be ineffective at mobilizing Ca2⫹ if it
never opened (gated). Conversely, a single RyR channel
with optimal gating behavior would be worthless if it did
not allow Ca2⫹ to pass efficiently. The opening and closing of the RyR channel presumably involves the physical
motion of the protein. Ions are able to traverse its pore
only when the channel is in the open configuration. It is
generally assumed that movement of the gate does not
alter the nature of the pore (i.e., its diameter, length,
charge, etc.). It is in the pore where the channel discrimination between different ions occurs. The pore also contains determinants that define how quickly particular ions
will pass. Specific RyR channel permeation properties are
described below. Specific features of single RyR channel
gating are discussed in later sections.
The permeation properties of the different RyR channel isoforms are quite similar. The RyR channels are
permeable to many different divalent and monovalent
cations (e.g., Refs. 167, 294, 330). In symmetrical solutions
Physiol Rev • VOL
containing a monovalent cation (e.g., ⬃200 mM K⫹, Na⫹,
or Cs⫹) as the main permeant species, the unitary RyR
channel current-voltage relationship is linear with a slope
conductance usually ⬎500 pS (167, 294). In asymmetric
solutions containing a divalent cation (e.g., ⬃50 mM Ca2⫹,
Ba2⫹, or Sr2⫹) as the main permeant species, unitary RyR
channel current-voltage relationships have a slope conductance of ⬃100 pS (330). These are very large conductance values. For comparison, high-conductance ion
channels of the plasma membrane such as the Ca2⫹activated K⫹ channels (i.e., maxi-KCa channels) have a
maximal conductance of near 250 pS (157). The unitary
Ca2⫹ conductance of the L-type Ca2⫹ channel (i.e., DHPR)
in the surface membrane is no more than ⬃20 pS (119).
Thus the RyR channels are very-large-conductance Ca2⫹
channels. This feature is probably fundamental to its
physiological role in cells, passing a large number of Ca2⫹
quickly.
The relative permeability of the RyR channels to
different monovalent and divalent cations has been defined under bi-ionic conditions (e.g., Refs. 167, 294, 330).
The RyR channels show very little selectivity between
different monovalent cations. The RyR channels also
show very little selectivity between different divalent cations. However, the RyR channels are selective, albeit
poorly, between mono- and divalent cations (PCa/PK ⬃6;
more permeable to Ca2⫹ than K⫹). This modest selectivity
for divalents is also unusual for a Ca2⫹ channel. The
typical surface membrane Ca2⫹ channel (e.g., DHPR) displays a much higher degree of discrimination between
monovalent and divalent cations (PCa/PK ⬎20). The point
is that the RyR channel is not a highly selective Ca2⫹
channel. Intuitively, the apparent deficiency in Ca2⫹ selectivity may be related to its high conductance. A highconductance channel (i.e., ions passing rapidly) would
have little time per ion to perform the steps needed to
discriminate between ions.
The relatively poor Ca2⫹ selectivity of the RyR channels does have important physiological ramifications. In
cells, Ca2⫹ is not the only ion that can permeate through
the open RyR channel pore. Other permeable cations
(Mg2⫹, Na⫹, and K⫹) are also present at substantial levels
(i.e., ⬃1, 10, and 140 mM, respectively). Thus Ca2⫹ competes with Mg2⫹, Na⫹, and K⫹ for occupancy of the RyR
channel pore. The consequence is that the unitary Ca2⫹
current carried by a single RyR channel in the cell (i.e., in
the presence of multiple permeant ions) will be considerably less than measured under the traditional simple recording conditions used in bilayer studies (e.g., Refs. 256,
259, 295, 330).
Tinker et al. (329) were the first to examine how
unitary Ca2⫹ current through the RyR2 channel is affected
by the presence of other permeant ions. They estimated
that unitary Ca2⫹ current (at 0 mV) under physiological
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In our experience, fusion of SR vesicles into planar
bilayers results in more stable/consistent single RyR
channel behavior compared with that obtained using
CHAPS-solubilized RyR channel preparations. As mentioned above, the use of SR vesicles requires that certain
specific steps be taken to avoid contaminating currents
from other ion channels. The first unitary Ca2⫹ current
recordings associated with the RyR channel were made
after heavy SR vesicles were fused in a bilayer in the
presence of ⬃50 mM Ca2⫹ and little else (291). The two
most common single RyR channel recording conditions
are listed below. One consists of a Ca-HEPES solution (50
mM Ca2⫹) on the luminal side with an isosmotic TrisHEPES solution on the cytosolic side of the channel. The
second consists of a Cs-methanesulfonate solution on
both sides of the channel. In both cases, contaminating
anion currents are avoided by the use of impermeable or
poorly permeable anions. In the first case, contaminating
cation currents are avoided by the presence of a nonpermeable organic monovalent cation (Tris⫹). In the second
case, contaminating K⫹ currents are avoided by using Cs⫹
as a permeant monovalent cation. The use of Cs⫹ works
because SR K⫹ channels are either poorly permeable to
Cs⫹ or blocked by this ion, while RyR channels are highly
permeable to Cs⫹ (as well as other monovalent cations).
901
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MICHAEL FILL AND JULIO A. COPELLO
duces very-long-duration open events and simultaneously
reduces ion conductance through the pore (e.g., Refs. 259,
294). This dual impact on single-channel function (i.e.,
slower gating, reduced conductance) suggests a complicated mode of action. This is also evidenced by the rather
complicated ryanodine dose dependency. Low doses of
ryanodine (⬃10 nM) are reported to increase the frequency of single RyR channel openings to the normal
conductance level (34). Intermediate ryanodine doses
(⬃1 ␮M) are reported to induce the classical ryanodine
action described above (34, 259, 294). High doses of ryanodine (⬃100 ␮M) are reported to lock the channel in a
closed configuration (368).
The fact that ryanodine binding alters single-channel
conductance suggests that ryanodine binds in or near the
RyR channel pore. In fact, the ryanodine-binding site has
been experimentally localized to the COOH terminus of
the protein (37), the region of the RyR protein thought to
contain the structural determinants of the pore. Altered
conductance suggests that ryanodine binding somehow
changes the nature of the permeation pathway. This idea
has also been experimentally confirmed. Two independent studies have indicated that ryanodine binding actually changes the effective diameter of the RyR channel
pore (332, 337).
The complex action of ryanodine may be related to
how the alkaloid binds to the channel. It is generally
assumed that each RyR monomer contains one high-affinity ryanodine binding site (KD ⬍50 nM; Refs. 147, 225). It
is possible that cooperative interaction between these
different ryanodine binding sites is what generates the
observed complexity (53, 242). Some ryanodine binding
studies suggest the existence of additional lower (KD ⬎1
␮M) affinity ryanodine binding sites (53, 147, 225, 242).
Thus the RyR channel complex may contain multiple
ryanodine binding sites. One interpretation suggests that
ryanodine binding to the high-affinity site depends on
whether or not the RyR channel is in the open configuration (53). It may be that conformational changes related
to the opening of the RyR channel exposes (or improves
access to) the high-affinity ryanodine binding site. This
would impart a use dependence to the kinetics of ryanodine binding, allowing “nonequilibrium” ryanodine binding to be used as a monitor of RyR channel function (as
described previously).
IX. OVERVIEW OF RYANODINE
RECEPTOR REGULATION
VIII. ACTION OF RYANODINE
The alkaloid ryanodine binds with high affinity to the
RyR proteins (225). Its binding induces a complex change
in single RyR channel function. This change is similar in
all three RyR channel isoforms. Ryanodine binding inPhysiol Rev • VOL
The RyR channels are modulated by numerous factors, including a number of physiological agents (e.g.,
Ca2⫹, ATP, and Mg2⫹), various cellular processes (e.g.,
phosphorylation, oxidation, etc.), and several pharmacological agents (e.g., ryanodine, caffeine, and ruthenium
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conditions would be ⬃1.4 pA. This still quite large current
was explained using a Eyring-rate model (328) assuming
that some mechanism allows the association rate of Ca2⫹
at the mouth of the pore to exceed, by about an order of
magnitude, the normal diffusional limit. A more recent
examination of unitary Ca2⫹ current through the single
RyR2 channel suggests that unitary Ca2⫹ current (at 0
mV) under physiological conditions may be closer to 0.5
pA (201). This is important because accurately defining
the amplitude of the unitary Ca2⫹ current through a single
RyR channel is fundamental to understanding the origin
of local intracellular Ca2⫹ signaling events (e.g., Ca2⫹
sparks) in cells. The unitary RyR Ca2⫹ current value is
central to the debate whether Ca2⫹ sparks arise from the
opening of one RyR channel or the concerted opening of
many RyR channels.
The larger unitary RyR channel Ca2⫹ current prediction suggests that a mechanism to enhance permeation
may possibly exist (328). The smaller unitary Ca2⫹ current prediction indicates that perhaps there is no need for
any permeation enhancement mechanism (201). What
might the permeation enhancement mechanism be? Several possible permeability enhancement mechanisms
have been proposed. It has been argued that the RyR2
channel may contain a large charged vestibule that dielectrically focuses ions near the pore’s mouth (328). Alternatively, dielectric focusing by fixed surface charges
could occur near the RyR channel’s mouth in the absence
of a large vestibule structure (337). It has also been suggested that a single RyR channel contains multiple parallel conduction pathways (i.e., a multibarrel pore). The
later possibility was supported by the initial 3-D RyR
channel reconstructions that suggested the presence of
multiple conduction pathways (342). Furthermore, Ondrias et al. (228) suggested that the four commonly observed subconductance states of the RyR channel might
correspond to uncoordinated opening of pores in the
individual RyR monomers of the tetrameric channel complex. They suggested that the presence of the FK-binding
protein synchronizes the openings of these separate permeation pathways. However, recent and more detailed
3-D reconstruction data suggest that the RyR channel
structure contains a single central pore (i.e., the RyR is
not a multibarrel channel; Refs. 230, 270, 275). However,
the possibility that the channel contains a large vestibule
and/or surface charges that impact ion permeation still
exists and should not be discounted.
RYANODINE RECEPTOR Ca2⫹ REGULATION
903
red). Some of these modulatory factors are discussed
below.
regulation). Distinguishing between these possibilities is
the challenge.
A. Cytosolic Ca2ⴙ
C. ATP and Mg2ⴙ
B. Luminal Ca2ⴙ
Early Ca2⫹ release studies suggested that Ca2⫹ binding to the luminal surface of the RyR channel may regulate the channel (64, 65). Single RyR channel studies have
confirmed the existence of luminal Ca2⫹ regulation (51,
110, 286, 285, 333, 341). It appears that the sensitivity of
RyR channel to certain cytosolic agonists increases at
high luminal Ca2⫹ levels (110, 286). Trypsin digestion of
the luminal side of the RyR channel suggested the presence of both activating and inactivating divalent sites
(51). It is possible that the luminal Ca2⫹ effects are mediated, at least in part, by associated proteins like calsequestrin (14, 314) or junctin (364).
It was also reported that luminal Ca2⫹ moving
through the channel feeds back and interacts with cytosolic Ca2⫹ binding sites (333). Thus luminal Ca2⫹ levels
may affect single RyR channel function by interacting
with luminal binding sites, with associated luminal proteins, or with cytosolic binding sites (i.e., feed-through
Physiol Rev • VOL
Cytosolic ATP is an effective RyR channel activator
(57, 60, 291, 297, 298, 353). Cytosolic Mg2⫹ is a potent RyR
channel inhibitor (57, 60, 158, 291, 353). The cytosol of
most cells contains ⬃5 mM total ATP and ⬃1 mM free
Mg2⫹. This means that most ATP is in its Mg2⫹-bound
form. Free ATP (⬃300 ␮M in the cytosol) is the species
that binds to and activates the RyR channel (297, 298).
The action of ATP and Mg2⫹ on single RyR channel function is isoform specific. Free ATP is a much more effective activator of the RyR1 channel than the RyR2 channel
(57, 60, 291, 297, 353). The RyR3 channel is also less ATP
sensitive than the RyR1 channel (46, 135, 297). The Mg2⫹
action on single RyR channels is complicated (57, 158).
First, Mg2⫹ may compete with Ca2⫹ at the Ca2⫹ activation
site, shifting the Ca2⫹ sensitivity of the channel (57, 60,
158, 353). Second, Mg2⫹ competes with Ca2⫹ at the high
Ca2⫹ inhibition site (57, 158). The high Ca2⫹ inhibition site
does not discriminate between different divalent cations.
The RyR1 channel is substantially more sensitive to this
action of Mg2⫹ than the RyR2 or RyR3 channels (57, 135).
In the presence of physiological levels of Mg2⫹ and ATP,
the RyR1 channel requires less Ca2⫹ to activate than the
RyR2 or RyR3 channel (57, 257, 320, 353).
D. Redox Status
Various redox processes may also modulate single
RyR channels. The RyR monomers contain 80 –100 cysteine residues (315a, 318). Many of these are suitable for
modification by oxidants. There is evidence that oxidant
and reducing agents do indeed impact single RyR channel
function (182, 222, 349), including the channel’s Mg2⫹
sensitivity. There is also evidence that oxidants may prevent certain closely associated regulatory molecules, like
calmodulin, to bind to the RyR channel (363). Recently, it
has been suggested that nitric oxide (NO) may modulate
the redox regulation of the RyR channels (79, 118, 352). It
was proposed that in vivo initial NO release may activate
RyR channels and subsequent increases in NO levels inhibit RyR channel activity (118). Thus there is a growing
interest in identifying the cysteine residues that may be
involved in RyR channel oxidation and/or S-nitrosylation
(352). It is quite possible that redox modulation of single
RyR channels represents an important regulatory mechanism. It is clear that additional information is needed in
this area.
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The action of Ca2⫹ on the RyR channel is complex.
The Ca2⫹ turns on, turns off, and conducts through this
channel. Steady-state single RyR channel activity is generally a bell-shaped function of cytosolic Ca2⫹ concentration (35, 46, 54, 56, 60, 135, 161, 224, 238, 285, 294, 333).
The RyR channels are activated by low Ca2⫹ concentrations (1–10 ␮M) and inhibited by high Ca2⫹ concentrations (1–10 mM). It has been reported that individual
RyR1 channels do not all respond in the same way (i.e.,
they are functionally heterogeneous) to activating cytosolic Ca2⫹ levels (56, 161, 178, 238). In contrast, individual
RyR2 and RyR3 channels respond much more homogeneously to cytosolic Ca2⫹ (46, 54, 56, 135, 161). Inhibition
at high cytosolic Ca2⫹ concentrations of the RyR1 is also
different from that of RyR2 and RyR3 channels. The RyR1
channel is almost entirely inhibited by 1 mM Ca2⫹,
whereas the RyR2 and RyR3 channels are not (35, 46, 54,
56, 60, 135, 161, 224, 225, 238, 264). Substantially higher
Ca2⫹ levels are required to inhibit the RyR2 and RyR3
channels (46, 54, 56, 135, 161). The reported half-maximal
inhibiting concentrations for the RyR2 and RyR3 channels
range from 2 to ⬎10 mM. It is not clear that such high
cytoplasmic Ca2⫹ concentrations are ever reached in the
cells. Thus the physiological role of this very high Ca2⫹
inhibition is not yet clear. Nevertheless, high Ca2⫹ inhibition of the RyR2 channels remains a controversial topic.
This is further discussed in section XV.
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MICHAEL FILL AND JULIO A. COPELLO
X. REGULATION BY CLOSELY
ASSOCIATED PROTEINS
E. Cyclic ADP-ribose
F. Phosphorylation/dephosphorylation
Analysis of the RyR protein sequence reveals that it
contains many consensus phosphorylation sites (315a,
318). For more than a decade, the effects of exogenously
applied kinases and phosphatases on single RyR channel
behavior have been examined (60, 114, 170, 189, 298, 315,
339, 348). The capacity of protein kinase A (PKA) to
activate the RyR1 channel has been reported (60, 298).
This is consistent with the observation that PKA significantly increases depolarization-induced Ca2⫹ release
from skeletal muscle SR vesicles (125). However, studies
in skinned and intact skeletal muscle fibers have not
supported this idea (27). The capacity of calmodulin
kinase and PKA to activate the RyR2 channel has also
been studied. Contradictory results have been reported
for calmodulin kinase (70, 114, 170). Similarly, some reports on PKA action find activation of RyR2 channels
(114), whereas others report a slight decrease in the
steady-state Po of RyR2 channels (339). Still others suggest PKA destabilizes RyR2 channel openings (i.e., promote substating) via phosphorylation-induced dissociation of FKBP12.6 (189). Yet another report shows that
PKA phosphorylation does not impact the RyR-mediated
Ca2⫹ spark in intact cardiac myocytes (166). The reason
for the appearance of such contradictory and confusing
results is unclear. It is possible that different experimental conditions are responsible. It is safe to conclude that
the impact of phosphorylation and/or dephosphorylation
on single RyR channel behavior will be debated for some
time.
Physiol Rev • VOL
A number of different proteins may be associated
with and modulate the RyR channels (60, 63, 136, 142, 144,
169, 181, 183, 185, 196, 231, 257, 287, 334, 343). However,
only a few of these proteins have been thoroughly studied
at the single-channel level. These few “well-studied” proteins include calmodulin, calsequestrin, FK-506 binding
protein, and certain fragments of the DHPR. A brief survey of how these proteins may interact with the RyR
channel is provided below.
A. Calmodulin
Calmodulin (CaM) was the first peptide found to
interact with single RyR channels in lipid bilayers (295).
CaM appears to bind the RyR in stoichiometric proportions (11, 207, 334). It binds to sites in the RyR channel
cytoplasmic assembly that are ⬃10 nm from the putative
entrance to the transmembrane pore (343). Application of
CaM can either activate (at low Ca2⫹ levels) or inhibit (at
high Ca2⫹ levels) the RyR1 and RyR3 channels (46, 99,
295, 334). For the RyR2 channel, only inhibitory effects of
CaM have been reported (99, 295). It was reported that
oxidation modifies RyR1 channel behavior and perhaps
its interaction with CaM (117, 363). It has also been
suggested that CaM may somehow protect the RyR1 channel from oxidative modifications during periods of oxidative stress (363). CaM also appears to bind to the DHPR
and consequently has also been implicated in the DHPRRyR1 interaction (117, 269). A potential role of CaM in
modulation of the RyR2 channel during E-C coupling is
not yet clear.
B. Calsequestrin
Calsequestrin is the main Ca2⫹ binding protein inside
the SR. Calsequestrin has a large number of acidic amino
acids that permit it to coordinately bind 40 –50 Ca2⫹/
molecule (181, 358). Calsequestrin is also known to aggregate in the presence of divalent cations (190). Electron
microscopy studies suggest that calsequestrin aggregates
are present in the regions of the SR (i.e., terminal cisternae) known to contain RyR channels (96). It has been
suggested that Ca2⫹- and pH-dependent conformational
changes in calsequestrin may modulate RyR channel activity (61, 120). It also has been suggested that calsequestrin action on the RyR channel may require the presence
of triadin, another RyR-associated protein (365). Thus
there is disagreement concerning the nature of the calsequestrin-RyR interaction.
Some studies in lipid bilayers suggest that calsequestrin activates the RyR channel (139, 226, 314). However,
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Cyclic ADP-ribose (cADPR) appears to play an important role in many nonmuscle systems (164). The
cADPR molecule, a metabolite of NADP, has been found
to act as a powerful intracellular Ca2⫹ release agent in sea
urchin eggs, and its action is thought to be mediated by
RyR or RyR-like channels (164). The direct action of
cADPR on RyR channels from muscle systems is controversial. An early report indicated that cADPR dramatically
activates single mammalian RyR channels (203). However, subsequent studies reported only minor effects of
cADPR (284) or no impact of cADPR at all (58, 100, 209).
The controversial reports may suggest a requirement of
an unrecognized cofactor and/or a potential interaction
with some closely associated regulatory factor (i.e., calmodulin, FK-506 binding protein; Refs. 58, 164). When
studied in intact cellular systems (that contain endogenous calmodulin and FK-binding proteins), cADPR had
only indirect action on RyR channel function (107, 126,
175). It appeared that cADPR activated the Ca2⫹-ATPase
(175), which indirectly activated the RyR channel by increasing luminal SR Ca2⫹ levels.
RYANODINE RECEPTOR Ca2⫹ REGULATION
one study suggests calsequestrin inhibits the RyR channel
(14). Thus the physiological ramifications of the calsequestrin-RyR interaction require clarification. Similarly,
the impact of calsequestrin in E-C coupling is unclear.
Studies in calsequestrin-deficient Caenorhabditis elegans
found no defects in body wall formation or locomotion,
suggesting calsequestrin is not essential for muscle contraction (52). On the other hand, overexpression of calsequestrin in mouse heart results in the development of
cardiac hypertrophy and altered Ca2⫹ signaling (143).
C. FK-506 Binding Protein
Physiol Rev • VOL
yet understood how the same protein (FKBP) could both
stabilize RyR monomer interactions within the RyR channel complex and synchronize the activity of neighboring
RyR channel complexes.
FKBP modulation of the RyR1 channel is generally
accepted (248), yet there is some uncertainty as to the
extent of this modulatory action. Studies in mechanically
skinned skeletal muscle fibers reported that FKBP12 removal reduces DICR or “uncouples” the DHPR-RyR1 interaction (155). The suggestion is that FKBP12 may be the
physical coupler between the DHPR voltage sensor and
the RyR1 channel. One study in human skeletal muscle
strips found that FKBP increases the contractile sensitivity to halothane and/or caffeine (33). Interestingly, animals deficient in FKBP12 die at embryonic stages (278),
attesting to the importance of FKBP. These animals die of
severe structural and functional abnormalities of the
brain and heart, but the characteristics of their skeletal
muscle are normal (278). This may imply that FKBP may
not be central to skeletal muscle structure-function. Furthermore, no apparent major defects in ambulation, or
other motor functions, were reported in animals with a
skeletal muscle-specific FKBP12 knockout (324). These
latter animals, however, had abnormal muscle contraction (decrease in contractility). Thus it appears that
FKBP12 does have some role in modulating RyR1-mediated Ca2⫹ release or muscle structure integrity (324). This
role, however, needs further clarification.
There are reports that suggest that FKBP12.6 is critical to normal RyR2 channel operation in heart (137, 192,
306). It has even been suggested that abnormal FKBP12.6RyR2 interactions may be implicated in certain types of
heart failure (184, 189, 229, 359). However, other studies
suggest that FKBP12.6 does not modulate the activity of
single RyR2 channels (12, 59, 327) or Ca2⫹ release in
isolated cardiac myocytes (68). Local RyR-mediated Ca2⫹
sparks in FKBP12.6 knockout mice have slightly increased amplitude and duration compared with those in
wild-type animals (351). In contrast, a previous report
showed that FK-506 or rapamycin (drugs that dissociate
FKBP12.6 from the RyR2) reduced Ca2⫹ spark amplitude
and increased spark duration by ⬃500% (350). The hearts
of male, not female, FKBP12.6 knockout mice develop
mild cardiac hypertrophy over time (351). However, the
hearts of these male FKBP12.6 knockout mice do not
progress to more severe myopathies or heart failure. This
implies that decreased cytosolic FKBP12.6 level, per se,
may not be the primary culprit in generation of these
pathologies. Thus the impact of FKBP12.6 (or FKBP12) on
single RyR2 function is not yet clearly established.
D. DHPR-Loop Peptide
Another protein that interacts with and regulates the
function of single RyR channels is the DHPR (323). As
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The FKBP12 and FKBP12.6 proteins (185) associate
with the RyR1, RyR2, and RyR3 proteins in apparently
stoichiometric proportions (326, 327). Thus there are four
FKBPs bound to each RyR channel complex. The RyR1
and RyR3 channels bind both the FKBP12 and FKBP12.6
forms. In cells, these RyR channels will have FKBP12
bound because of its high abundance in the cytosol (185,
326, 327). The RyR2 channel in dog heart appears to
preferentially bind FKBP12.6 (327), and thus RyR2 channels will have FKBP12.6 bound to them. The RyR2 channels in other species have slightly less specificity for
FKBP12.6 and thus may be bound to both FKBP12.6 and
FKBP12 (134).
How does FKBP binding impact single RyR channel
function? There is a good consensus that removal of
FKBP12 from the RyR1 channel activates the channel (3,
12, 32, 48, 191, 326). The FKBP impact on RyR2 channel
function is not so clear. Some groups report that removal
of FKBP12.6 from the RyR2 channel activates the channel
(137, 350). However, other groups report that FKBP12.6
removal has no impact on RyR2 channel function (12, 59,
327, 351). Thus there is disagreement concerning the action of the FKBPs on RyR2 channel function. Some points
of this controversy are described below.
Several groups have shown that FKBP removal promotes the appearance of subconductance states (3, 4, 32,
137, 189). Subconductance substates are open events with
less than normal unit current amplitude. This observation
led to the suggestion that FKBP binding stabilizes the RyR
channel complex and in doing so stabilizes the structure
of the permeation pathway. Some groups, however, do
not report the appearance of subconductance substates in
FKBP12.6-depleted RyR2 channels or RyR2 channels isolated from FKBP12.6-deficient mice (12, 59, 327). Subconductance states are also only rarely observed in “purified”
RyR1, RyR2, and RyR3 channels (135, 211, 352) known to
be largely devoid of FKBP (211, 343). More recently, it has
been suggested that FKBP may synchronize the singlechannel function neighboring RyRs (188). The suggestion
is that this synchronization permits several neighboring
RyR channels to open and close simultaneously. It is not
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MICHAEL FILL AND JULIO A. COPELLO
XI. CALCIUM REGULATION PARADOX
Although the RyR channels are modulated by many
factors, Ca2⫹ regulation plays a central role in all RyRmediated signaling cascades. In some cases (i.e., cardiac
muscle), the regulating Ca2⫹ signal initiates RyR channel
activity. In other cases (i.e., skeletal muscle), the regulating Ca2⫹ signal may amplify RyR channel activity. There is
also evidence that regulating Ca2⫹ signals may terminate
RyR channel activity. The importance of understanding
how Ca2⫹ regulates the RyR channels cannot be overstated.
Our discussion of RyR2 channel Ca2⫹ regulation
starts with the classical work of Fabiato (80, 81), which
Physiol Rev • VOL
defined the Ca2⫹ regulation of the SR Ca2⫹ release process in a skinned cardiac muscle preparation. This work
showed that the amplitude and duration of trigger Ca2⫹
stimulus finely grades the CICR process (i.e., Ca2⫹-induced Ca2⫹ release). Specifically, the amount of Ca2⫹
released from the SR was a bell-shaped function of trigger
Ca2⫹ stimulus amplitude, and that trigger Ca2⫹ stimulus
speed scaled this relationship.
Intuitively, the CICR process should be self-regenerating because the Ca2⫹ released by a RyR2 channel should
feedback and further activate the channel or its neighbors. However, self-regeneration of CICR is not observed
in cells. This is the Ca2⫹ regulation paradox. The implication is that some sort of negative-feedback mechanism(s) must exist to counter the inherent positive feedback of CICR. Fabiato (81) proposed that the negativefeedback mechanism was Ca2⫹-dependent inactivation.
The idea was that two different Ca2⫹ binding sites (activating and inhibitory, respectively) regulate the Ca2⫹ release channel (i.e., RyR2). The Ca2⫹ activation process
was thought to have a fast action and a relatively low
affinity. The Ca2⫹ inactivation process had a slower action but a relatively high Ca2⫹ affinity (81). Consequently,
a fast Ca2⫹ stimulus transiently activates the channel
because the channel would be “turned on” as Ca2⫹ occupies the activation site and then “turned off” as Ca2⫹ more
slowly acts at the inactivation site. A slow Ca2⫹ stimulus
would not effectively activate the channel because inactivation would “keep pace” with the activation process. At
very high Ca2⫹ levels (⬎100 ␮M), the inactivation sites
would be saturated, leaving the channel in an inactivated
state. The mechanism of Ca2⫹-dependent inactivation as
envisioned by Fabiato (81) implies that the channel becomes refractory (unable to respond to further Ca2⫹ stimulation). Recovery from the refractory state would require
removal of the Ca2⫹ stimulus and enough time for some
sort of recovery process (i.e., repriming) to occur. This
scheme is still widely discussed and is often applied to
explain the complex Ca2⫹ regulation of many RyR-mediated Ca2⫹ signaling events.
Despite its popularity, the existence of high-affinity
Ca2⫹-dependent inactivation has been challenged. Studies
on patch-clamped cardiac myocytes have not conclusively confirmed the existence of the Ca2⫹-dependent
inactivation phenomenon (208, 214, 360). For example,
the capacity of a second Ca2⫹ stimulus to trigger SR Ca2⫹
release was shown to be independent of the interval
between the first and second stimuli (214). The argument
here is that if the SR Ca2⫹ release channels were inactivated after the first stimulus, then the effectiveness of the
second stimulus should be affected. In another study
(360), a prolonged triggering Ca2⫹ signal was used to
inactivate the Ca2⫹ release process. A subsequent trigger
Ca2⫹ signal, however, reactivated the apparently inactivated release process (i.e., the process was not refractory).
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described previously, the DHPR-RyR interaction is clearly
involved in the signaling that links surface membrane
excitation to SR Ca2⫹ release in striated muscle. The
nature of the DHPR-RyR interaction is tissue specific. In
skeletal muscle, the interaction is thought to involve a
direct physical link between the two proteins. In cardiac
muscle, the DHPR Ca2⫹ channel mediates a small Ca2⫹
influx that activates the RyR2 channel. Our attention here
will be focused on the physical DHPR-RyR1 link in skeletal muscle.
Tanabe et al. (321) showed that the cytoplasmic II-III
loop of the skeletal DHPR ␣1-subunit is required for the
functional DHPR-RyR1 interaction. Although other regions of the DHPR may be involved (62, 165, 289), the
potential role of the II-III loop has been studied most to
date. Peptide fragments that correspond to the II-III loop
activate single RyR1 channels in planar lipid bilayers (171,
172). Interestingly, different regions of the peptide appear
to have different actions on RyR1 channel function. One
region called peptide A activates single RyR1 channel (71,
171, 172, 262). Another region called peptide C blocks the
activating action of peptide A (71, 171, 262). Additional
insights have been gained from examining the action of
the scorpion peptide toxin imperatoxin A, whose structure has similarities to that of peptide A (108). Like peptide A, imperatoxin A activates single RyR1 channel in
planar bilayers (108). The toxin binds with high affinity to
the RyR1 channel but at a site that appears to be quite
distant from the RyR1 regions thought to interact with the
DHPR (263). If peptide A and imperatoxin A bind to the
same site, then the distant location of this site is difficult
to reconcile with our current understanding of the DHPRRyR1 interaction. Recently, the interpretation of the physiological role of peptide A on RyR1 channel function was
further complicated. Grabner et al. (106) showed that a
chimeric DHPR, lacking the peptide A region, supported
normal DHPR-RyR1 signaling when expressed in dysgenic
myotubes (i.e., myotubes lacking endogenous DHPR).
Thus the role of peptide A in the DHPR-RyR1 interaction
has yet to be clearly established.
RYANODINE RECEPTOR Ca2⫹ REGULATION
Still, it is possible that the second Ca2⫹ stimulus (in both
cases above) was activating neighboring Ca2⫹ release
sites that did not participate in the first Ca2⫹ stimulus. In
any event, studies at the whole cell level have not conclusively confirmed or dismissed the existence of Ca2⫹dependent inactivation. Studies of RyR2 channel Ca2⫹
regulation at the single-channel level promised to resolve
this question.
XII. STEADY-STATE CALCIUM DEPENDENCE
XIII. CALCIUM ACTIVATION KINETICS
Several groups have defined the kinetics of single
RyR2 channel Ca2⫹ regulation in bilayers (e.g., Refs. 112,
113, 159, 264, 283, 339). There is considerably less data
Physiol Rev • VOL
addressing the kinetics of single RyR1 channels (e.g., Ref.
340) and none concerning single RyR3 channel function.
Consequently, the discussion here focuses mainly on the
kinetics of RyR2 channel Ca2⫹ regulation.
A. Rate of Ca2ⴙ Activation
Fast Ca2⫹ stimuli were applied to single RyR2 channels using two different methodologies. One methodology
generates fast Ca2⫹ stimuli by liberating Ca2⫹ from cagedCa2⫹ compounds using laser flash photolysis (112, 339,
340). The other methodology generates fast Ca2⫹ stimuli
by mechanically changing the solution near the RyR2
channel (159, 264, 283). The RyR2 channel activates with
a time constant of ⬃1 ms when the Ca2⫹ is very rapidly
changed via flash photolysis of caged-Ca2⫹ and slightly
slower (␶ ⫽ 2–20 ms) when Ca2⫹ is changed by a mechanical solution change technique. These numbers are fairly
consistent and are generally in agreement with whole cell
studies that clearly show that Ca2⫹ activation of SR Ca2⫹
release occurs on a millisecond time scale.
Interestingly, the reported RyR2 channel Ca2⫹ activation time constants vary with the speed of the applied
Ca2⫹ stimulus. For example, Laver and Curtis (159) applied relatively slow mechanical Ca2⫹ changes and reported a time constant of ⬍20 ms. Sitsapesan et al. (283)
applied slightly faster mechanical Ca2⫹ changes and reported a Ca2⫹ activation time constant of ⬍10 ms.
Schiefer et al. (264) applied even faster mechanical Ca2⫹
changes and reported a time constant between 2 and 5
ms. The fastest Ca2⫹ changes, however, were applied
using the flash photolysis method. These studies report a
Ca2⫹ activation time constant of ⬃1 ms (112, 113, 339).
The interpretation of the mechanical solution change
data described above must consider the presence of unstirred layers immediately in front of the bilayer. In some
of the mechanical solution change studies (159, 283), the
size of the unstirred layers clearly impacted the rate of
Ca2⫹ change in the microenvironment of the channel.
Unfortunately, concerns about the impact of unstirred
layers have rarely been discussed. Unstirred layers would
tend to slow the measured Ca2⫹ activation time constant.
Unstirred layers, however, do not complicate the interpretation of the flash photolysis data, but there is another
concern here. The Ca2⫹ waveform applied by flash photolysis is not a simple square step. Instead, there is a fast
Ca2⫹ overshoot at its leading edge (77, 78, 194, 372). This
very brief Ca2⫹ overshoot (lasting ⬃150 ␮s) likely accelerates the initial opening transition of the RyR2 channel.
The overshoot in these studies would tend to speed up the
measured Ca2⫹ activation time constant. Thus there are
studies (flash photolysis) that may overestimate the Ca2⫹
activation kinetics as well as studies (mechanical solution
change) that may underestimate it.
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As discussed above, several laboratories have defined the Ca2⫹ dependence of single RyR1 and RyR2
channels under steady-state conditions (e.g., Refs. 54, 56,
161, 256). In the absence of other modulating agents,
these studies show that there is little spontaneous channel activity at low cytosolic free Ca2⫹ concentrations
(⬍100 nM). This is consistent with the rarity of spontaneous Ca2⫹ release events (i.e., Ca2⫹ sparks) at resting
intracellular Ca2⫹ levels in cells (25, 50). There is substantial spontaneous activity of single RyR channels at steadystate Ca2⫹ concentrations in the micromolar range. The
degree of spontaneous activity peaks at ⬃10 ␮M. The
apparent KD of single RyR channel Ca2⫹ activation is
typically between 0.5 and 5 ␮M. This is consistent with the
Ca2⫹ sensitivity of SR Ca2⫹ release activation in skinned
cardiac cells (81) and isolated cardiac and skeletal SR
vesicle preparations (54, 199, 234 –236, 355).
The activity of single RyR1 channel decreases to near
zero if the steady-state Ca2⫹ concentration is elevated to
⬃1 mM (54, 56, 161, 238). This is not true for the RyR2 or
RyR3 channels. Substantial inhibition of these channels is
only observed when the steady-state Ca2⫹ concentration
exceed 5–10 mM (54, 56, 135, 161, 238). It is unlikely that
the free Ca2⫹ levels in the cytosol of a cell would ever
exceed the 1 mM mark because the Ca2⫹ sources used to
elevate cytosolic Ca2⫹ (i.e., extracellular solution and SR
lumen) contain only ⬃1 mM Ca2⫹. Thus the physiological
relevance of such low-affinity Ca2⫹ inhibition is questionable. It is unclear how such low affinity of Ca2⫹ inhibition
(as observed at the single-channel level) is related to the
apparent high-affinity Ca2⫹-dependent inactivation observed in cells (81). However, the RyR channels operate in
a dynamic (not stationary) Ca2⫹ signaling environment.
Thus steady-state measurements of channel activity (over
minutes) may not reveal all the intricacies of single RyR
channel Ca2⫹ regulation in cells (on a millisecond time
scale).
907
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MICHAEL FILL AND JULIO A. COPELLO
B. Rate of Ca2ⴙ Deactivation
Fast Ca2⫹ elevations clearly turn on single RyR2
channels rapidly (112, 264). An equally important question
is, How quickly do single RyR2 channels turn off (deactivate) in response to fast Ca2⫹ reductions? Intuitively, it
will take some time for Ca2⫹ to “fall off” the Ca2⫹ activation site and for the channel to move into a closed configuration. The Ca2⫹ deactivation kinetics of single RyR2
channels have been explored using both fast solution
exchanges (264, 283) and flash photolysis (340). Sitsapesan et al. (283) reported that the time constant of RyR2
channel Ca2⫹ deactivation was ⬍10 ms, about the measured rate of the Ca2⫹ change. Schiefer et al. (264) reported that RyR2 channel Ca2⫹ deactivation occurred
with a time constant of ⬃6 ms, ⬎10 times slower than
measured Ca2⫹ change. Velez et al. (340) photolyzed
Diazo-2 (a caged Ca2⫹ chelator) to reduce the local free
Ca2⫹ concentration near a single RyR2 channel in planar
bilayers. They reported a deactivation time constant of 5.3
ms, ⬎10 times slower than measured Ca2⫹ change. Thus
there is reasonable consensus that single RyR2 channels
deactivate rapidly with a time constant of 5– 6 ms in
response to a fast Ca2⫹ reduction.
The point is that the Ca2⫹ activation and deactivation
kinetics of single RyR2 channels in vitro are sufficient for
the channel to respond to the very brief (⬍1 ms) trigger
Physiol Rev • VOL
Ca2⫹ concentration changes, the kind of local trigger
Ca2⫹ signals that are thought to govern RyR2 channel
activity in vivo (302).
XIV. FEEDBACK CALCIUM REGULATION
One suggestion is that the Ca2⫹ interacting with the
Ca activation site is different from the Ca2⫹ that passes
through the open RyR channel pore. Another hypothesis
is that the Ca2⫹ activation site may “see” the Ca2⫹ flux
that passes through its own open pore. In other words, the
RyR channel may operate in a local “common Ca2⫹ pool”
(302). There are several whole cell observations that are
consistent with this latter common Ca2⫹ pool idea (25,
168, 237, 344).
Is this type of self-feedback Ca2⫹ regulation seen in
single RyR channel studies? Most single RyR studies have
been done under conditions that generate superphysiological unitary Ca2⫹ currents (285, 290 –292, 306). The
pertinent single RyR channel studies were done with huge
Ca2⫹ driving forces that are more than 50 times greater
than those thought to exist in cells. This would increase
the possibility of feedback Ca2⫹ regulation in ways that
do not exist in the cell. This fact must be considered here.
The steady-state cytosolic Ca2⫹ sensitivity of the
RyR1 or RyR2 channels appears to be the same whether
the charge carrier is Ca2⫹ or a monovalent cation (58, 161,
290, 294, 306). This observation implies, but does not
prove, that charge carrier identity does not impact RyR
channel Ca2⫹ regulation. Sitsapesan and Williams (285)
have evaluated single RyR2 channel gating in the presence of different luminal Ca2⫹ concentrations. They concluded that Po and open/closed lifetimes of Ca2⫹-activated, Ca2⫹-conducting RyR1 channels were independent
of the Ca2⫹ flux through the channel. In contrast, Tripathy
and Meissner (333) reported that the duration of single
RyR1 channel openings was significantly longer in the
presence of even a relatively small Ca2⫹ flux through the
pore. They go on to predict that the RyR1 channel’s Ca2⫹
activation site must be ⬃75 nm from the pore to explain
their results. Interestingly, the RyR channel complex is
only ⬃25 nm square (230, 249, 342). This may indicate that
the RyR1 channel’s Ca2⫹ activation site may be in some
sort of protected pocket that shields the site from the
Ca2⫹ coming through the pore. In any event, it is clear that
additional data are required to establish whether or not
feedback Ca2⫹ regulation occurs.
2⫹
XV. POTENTIAL NEGATIVE
CONTROL MECHANISMS
It is clear that some sort of RyR channel negative
control mechanism must exist to counter the inherent
positive feedback of the CICR process (81, 105, 214, 255,
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What is the real RyR2 channel Ca2⫹ activation kinetics? The single RyR2 channel Ca2⫹ activation time constant (␶ ⬃2–5 ms) to a steplike Ca2⫹ stimulus is probably
best represented by the data of Schiefer et al. (264). This
study applied very fast mechanical Ca2⫹ changes on very
small-diameter bilayers alleviating unstirred layer concerns. These types of experiments, however, have never
been repeated. The physiological Ca2⫹ trigger in cells is
clearly not a simple square Ca2⫹ step. The RyR2 channels
in vivo are governed by the very fast transient Ca2⫹
changes that occur near DHPR (i.e., L-type) Ca2⫹ channels when they flicker open (302). It has been estimated
that the free Ca2⫹ concentration near the open mouth of
a DHPR Ca2⫹ channel can reach very high levels (⬃100
␮M) when it is open and fall very rapidly (⬃10 ␮s) after
the channel closes. Interestingly, the fast Ca2⫹ overshoot
generated by flash photolysis has similar characteristics
(78). The rise time of the fast Ca2⫹ overshoot has a time
constant of ⬃30 ␮s, and the Ca2⫹ overshoot can theoretically peak at 50 –100 ␮M before it decays with a time
constant of ⬃150 ␮s. Thus the fast Ca2⫹ overshoot may be
a reasonable reproduction of the free Ca2⫹ changes that
initiate RyR2 channel activity in the cell. Therefore, the
single RyR2 channel Ca2⫹ activation time constant to
physiological Ca2⫹ stimuli may be best represented by the
flash photolysis data (␶ ⬃1 ms; Ref. 112).
RYANODINE RECEPTOR Ca2⫹ REGULATION
265, 302, 303, 307). There is a growing consensus that the
negative control that counters the inherent positive feedback of CICR is actually a composite of factors/processes
that act in concert to terminate local RyR-mediated Ca2⫹
release. This latter idea is consistent with the fact that no
individual negative control mechanism appears sufficient
by itself to explain the termination of local Ca2⫹ release.
Several candidate RyR channel negative control mechanisms are discussed below.
A. Ca2ⴙ-Dependent Inactivation
B. Stochastic Attrition
Stochastic attrition refers to the inherent random
closing of an individual ion channel. Stern (302) demonstrated using mathematical modeling that a SR Ca2⫹ release unit composed of one or relatively small number of
RyR2 channels will turn off spontaneously as a result of
reduced local Ca2⫹ levels resulting from the stochastic
closures of individual RyR2 channels in the cluster. Thus
Physiol Rev • VOL
stochastic attrition of single RyR2 channel activity could
contribute to terminating a local Ca2⫹ release event. This
process, however, would be very sensitive to the number
of channels involved and the positive feedback gain of
CICR in the channel cluster. Stern (302) showed that
stochastic attrition was sufficient to terminate local Ca2⫹
release only if there were ⬍10 RyR2 channels per cluster.
If release units are composed of 10 –30 or more channels
(31, 95, 174), then the likelihood of termination solely by
stochastic attrition is low. Experimentally, Lukyanenko et
al. (176) showed that the rate of Ca2⫹ release termination
is proportional to the magnitude of the release event (i.e.,
large events terminate faster than small events). Additionally, Sham et al. (273) demonstrated that local Ca2⫹ release events terminate rapidly despite the presence of a
sustained trigger Ca2⫹ signal. These results are inconsistent with stochastic attrition playing a major role in control of the CICR process. Nevertheless, stochastic attrition may still contribute to local Ca2⫹ release termination
if it acts in combination with other negative control mechanisms. It should be noted that the action of stochastic
attrition would be highly nonlinear because once the
number of active channels falls below some critical level,
the remaining channels would be increasingly impacted
by the phenomenon.
C. Adaptation
Most single RyR channels studies have focused on
defining RyR behavior under steady-state conditions.
Györke and Fill (112), using laser flash photolysis of
caged Ca2⫹, were the first to apply fast trigger Ca2⫹
signals to single RyR2 channels in bilayers. They reported
that single RyR2 channels activated rapidly reaching Po
values that were well above those predicted from previous steady-state studies. After an initial burst of singlechannel activity, Po spontaneously decayed with a time
constant of ⬃1 s. Application of second Ca2⫹ stimulus
reactivated the apparently “inactivated” channels. This
suggested that the spontaneous decay observed after the
first Ca2⫹ stimulus was not due to a conventional absorbing Ca2⫹ inactivation mechanism (81) because the channels were not in a refractory state. They proposed that the
spontaneous decay may be mediated by a different process they termed adaptation (112, 230).
The RyR2 channel adaptation proposal spurred an
active debate. One point of debate centered on the relatively slow time course of adaptation (⬃1 s). The point
was that adaptation might be too slow to be a negative
control mechanism that stabilizes the CICR process in
heart. Somewhat lessening this concern, Valdivia et al.
(339) demonstrated that the RyR2 channel adaptation
phenomenon was ⬃10-fold faster in the presence of physiological Mg2⫹. Another point of debate was that not all
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The first negative-feedback mechanism considered
was Ca2⫹-dependent inactivation (81). It was reported
that the SR Ca2⫹ release process is substantially inactivated at steady-state Ca2⫹ concentrations as low as 60 nM
in skinned cardiac muscle fibers (81). However, single
RyR2 channel studies in bilayers have forwarded no evidence of Ca2⫹-dependent inactivation at such low Ca2⫹
concentrations (54, 161, 256). Also, subsequent studies on
intact cells presented contradictory results (109, 208, 214,
280). For example, Lukyanenko and Györke et al. (173)
imaged permeabilized ventricular myocytes using a confocal microscope and showed that elevation of resting
Ca2⫹ levels resulted in an increase in RyR2-mediated Ca2⫹
spark activity, not the decrease expected if Ca2⫹-dependent inactivation exists. Thus the existence of high-affinity Ca2⫹-dependent inactivation is debated. The existence
of low-affinity Ca2⫹-dependent inactivation is not. Single
RyR1 and RyR2 channel data clearly demonstrate the
existence of low-affinity Ca2⫹ inhibition under steadystate experimental conditions. Single RyR1 channels
clearly turn off as the cytosolic free Ca2⫹ concentration is
elevated toward the 1 mM mark (196, 253, 307). This
low-affinity Ca2⫹ inhibition could come into play if local
Ca2⫹ levels in the cell reach the 0.6 –1 mM range (156).
For the RyR2 channel, the situation is different. Single
RyR2 channels turn off only after free Ca2⫹ concentrations reach much higher levels (5–10 mM; Refs. 12, 54, 56,
161, 199, 256). These levels are probably never realized in
a cell. Thus Ca2⫹-dependent inactivation as a potential
negative control mechanism that may account for (or
contribute to) the termination of local RyR-mediated
Ca2⫹ release events is still not clearly established.
909
910
MICHAEL FILL AND JULIO A. COPELLO
D. Activation-Dependent or “Fateful” Inactivation
Pizarro et al. (244) found that the global RyR1-mediated Ca2⫹ release transient in frog skeletal muscle, triggered by a maximally depolarizing stimulus, was inhibited
in proportion to the degree of Ca2⫹ release generated
during a prestimulation. This suggests that the RyR1 channels activated in the prestimulation were not available for
activation during the main stimulus. Thus preactivated
RyR1 channels appeared inactivated or refractory. These
authors proposed that RyR1 channel inactivation is
strictly and “fatefully” linked to their activation. In other
words, RyR1 channel inactivation is use dependent. Certain single-channel studies are consistent with this idea. It
is possible that a modal gating shift (i.e., high to low Po
modes) may be a single-channel manifestation of fateful
inactivation (i.e., use dependence) observed in situ. Several different groups have now reported modal gating
behavior of single RyR channels (8, 86, 111, 361).
E. Depletion/Luminal Ca2ⴙ Effects
When the SR is overloaded with Ca2⫹, periodic spontaneous Ca2⫹ waves can occur in heart muscle (304). It
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appears that luminal Ca2⫹ levels modulate these RyR2mediated Ca2⫹ waves. The presence of a luminal Ca2⫹
regulation may also explain why a critical Ca2⫹ load must
be attained before Ca2⫹ can trigger SR Ca2⫹ release from
SR vesicle preparations (234 –236). Several single RyR
channel studies have also addressed the possibility that a
luminal Ca2⫹ regulatory site exists (110, 341, 285, 333).
Velez and Suarez-Isla (341) showed that RyR channel
activity changed as a function of luminal Ca2⫹ concentration and suggested this phenomenon was mediated by a
low-affinity Ca2⫹ binding site on the luminal side of the
channel. Later, two groups (110, 285) systematically investigated luminal RyR2 Ca2⫹ regulation and showed that
its extent depended on how the RyR2 channel was activated. For example, ATP-activated RyR2 channels were
more sensitive to luminal Ca2⫹ levels than Ca2⫹-activated
RyR2 channels.
The pertinent low-affinity luminal Ca2⫹ binding
site(s) may reside on the RyR channel or possibly on
another closely associated luminal protein. These sites
would need to sense changes in intra-SR Ca2⫹ levels in the
appropriate Ca2⫹ concentration range (0.2–20 mM). As SR
Ca2⫹ levels fall, the RyR channel would sense the reduced
SR Ca2⫹ level leading to a decrease in channel activity.
This would then represent an interesting form of RyR
negative control. There are caveats, however, in this luminal Ca2⫹ story. Repetitive Ca2⫹ sparks should reduce
local SR Ca2⫹ load and thus downregulate RyR2 channel
activity. Yet, multiple Ca2⫹ sparks can occur frequently at
the same site in cells with little sign of progressive rundown. This may suggest that SR Ca2⫹ levels either do not
change dramatically and/or that luminal Ca2⫹ may not
impact RyR2 channel activity dramatically. The spark
data, however, do not rule out the possibility that very
brief and highly localized luminal Ca2⫹ depletions govern
RyR2 channel function.
F. Allosteric or Coupled Gating
Recently, Stern et al. (305) evaluated several published single RyR2 gating schemes and found that they all
generated unacceptable instability when applied in a
model of Ca2⫹ release local control. One reason for the
instability was the lack of a strong negative control mechanism to minimize local regenerative Ca2⫹ release in clusters of RyR2 channels. Another reason for the instability
was the relatively low cooperativity of the RyR2 channel
Ca2⫹ activation process. The low cooperativity does not
allow the RyR2 channels to adequately discriminate between trigger and background Ca2⫹ signals. Interestingly,
Stern et al. (305) suggested that RyR2-RyR2 allosteric
interactions could theoretically overcome these instability problems. Recently, it has been reported that neighboring RyR1 channels may be physically coupled by the
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investigators reported “adaptive behavior” of single RyR2
channels. Although several groups reported that fast Ca2⫹
stimuli induced a transient burst of single RyR2 channel
activity (112, 159, 264, 283, 339), different groups interpreted their results in different ways. Still another point of
debate was centered on the nature of the Ca2⫹ stimulus
applied in the original adaptation study (112). It was
suggested that the fast Ca2⫹ overshoot at the leading edge
of the applied Ca2⫹ stimulus might generate the observed
adaptation phenomenon (154). As a result, the single
RyR2 channel data generated by the flash photolysis have
undergone an unusual level of scrutiny. It has endured,
has been frequently reviewed (86, 152, 288), and has
generated new insights into single RyR2 channel Ca2⫹
regulation. Details concerning the RyR2 adaptation debate are discussed in section XVI.
We now know that the adaptation phenomenon is
due to a Ca2⫹- and time-dependent shift in the modal
gating behavior of single RyR2 channels (111). A fastsustained Ca2⫹ stimulus transiently drives the RyR2 channel into a high Po gating mode before it has time to
equilibrate (i.e., adapt) between all its different gating
modes. A specific kinetic scheme of RyR2 channel gating
has been developed describing this behavior (86). It is
now clear that Ca2⫹-dependent inactivation and adaptation are not mutually exclusive mechanisms (as generally
thought). These two phenomena may simply be different
manifestations of a common underlying mechanism,
Ca2⫹- and time-dependent modal gating.
RYANODINE RECEPTOR Ca2⫹ REGULATION
FK506-binding protein (FKBP12) and consequently gate
in synchrony (188). Is this coupled RyR channel gating
operative under physiological conditions? The answer to
this question is not yet clear (19, 305). Other investigators
have not reported coupled RyR channel gating. Additionally, certain thermodynamic considerations may also be a
problem (i.e., multiple large macromolecules operating
with microsecond synchrony). Furthermore, removal of
FKBP12.6 from the RyR channel does not abolish Ca2⫹
sparks, but actually increases their frequency and duration (351). Although coupled RyR channel gating is an
interesting and provocative hypothesis, its existence and
nature still require verification.
The Györke and Fill (112) RyR2 adaptation hypothesis was controversial. One concern was that the decrease
in RyR2 channel activity, interpreted as adaptation, might
reflect slow Ca2⫹ deactivation (154) following a shortlived Ca2⫹ transient (i.e., the fast Ca2⫹ overshoot) at the
leading edge of the applied Ca2⫹ stimuli. This possibility
was experimentally addressed, and the data suggest that
the brief Ca2⫹ overshoot has little impact on the much
slower (i.e., 1,000-fold slower) adaptation phenomenon.
The data indicate that the impact of the fast Ca2⫹ overshoot is most likely limited to accelerating the initial
closed to open transition of the RyR2 channel (i.e., it
essentially supercharges the applied Ca2⫹ stimuli). A detailed discussion of the Ca2⫹ overshoot concern is presented below after a detailed description of the Ca2⫹
overshoot itself. Another concern regarding the original
adaptation hypothesis (112) was that some investigators
did not report adaptive behavior of single RyR2 channels
but instead forwarded alternate interpretations (49, 112,
159, 264, 283, 339). Some of these different interpretations
are also presented below.
A. DM-nitrophen Ca2ⴙ Overshoot
DM-nitrophen is an ultraviolet-sensitive high-affinity
Ca2⫹ buffer that changes its Ca2⫹ affinity from ⬃4.8 nM to
⬃3 mM upon flash photolysis (74, 78, 194). Ca2⫹ liberation
by flash photolysis of DM-nitrophen (the caged Ca2⫹ compound used the RyR2 adaptation studies) generates a fast
Ca2⫹ overshoot (78, 154, 194, 372). It arises because flash
photolysis liberates Ca2⫹ faster than free DM-nitrophen
can bind Ca2⫹. Its characteristics and how its kinetics
vary with experimental conditions are not generally understood and often the basis of debate (112, 154, 372).
The fast Ca2⫹ overshoot is very fast compared with
kinetics of commonly used fluorescent dyes (78). Consequently, the kinetic characteristics of the fast Ca2⫹ overshoot are necessarily based on numerical calculations
Physiol Rev • VOL
from the much slower fluorescence data even in the case
of the fastest fluorescent indicators (78). The parameters
estimated for the fast Ca2⫹ overshoot may then reflect
more the kinetic characteristics of the indicator than the
Ca2⫹ liberation kinetics of the caged Ca2⫹ compound.
There is no debate that DM-nitrophen photolysis by a
brief flash (⬍50 ns long) liberates Ca2⫹ in ⬍30 ␮s (74, 78,
194). However, there is disagreement about the Ca2⫹
association rate of DM-nitrophen. Zucker (372) suggested
a relatively slow association rate (1.5 ⫻ 106 M⫺1䡠s⫺1), and
use of this value predicts that Ca2⫹ overshoots should last
several milliseconds. More recently, Escobar et al. (78)
showed that the association rate was substantially faster
(3 ⫻ 107 M⫺1䡠s⫺1). This means that the fast Ca2⫹ overshoot is more than an order of magnitude faster (⬃100 ␮s
vs. ⬃2 ms) than previously thought (154, 372).
The magnitude of the overshoot depends on the free
DM-nitrophen levels. At pCa 9, only ⬃18% of unphotolysed high-affinity DM-nitrophen will be bound to Ca2⫹.
Flash photolysis will liberate Ca2⫹ from Ca2⫹-bound DMnitrophen, but much of this Ca2⫹ will rebind to Ca2⫹-free
DM-nitrophen. At pCa 7, nearly 96% of unphotolysed highaffinity DM-nitrophen will be bound to Ca2⫹. The same
flash will liberate much more Ca2⫹ from Ca2⫹-bound DMnitrophen, but there will be less Ca2⫹-free DM-nitrophen
to participate in rebinding. As a result, the Ca2⫹ overshoot
will be smaller and slower. At pCa 6, nearly all unphotolysed high-affinity DM-nitrophen will be bound to Ca2⫹.
There will thus be no Ca2⫹ rebinding and consequently no
Ca2⫹ overshoot.
We estimate, based on the calculations presented in
previously works (78, 86), that the Ca2⫹ overshoot in the
original Györke and Fill (112) study peaked at 30 – 60 ␮M
and lasted ⬃100 –200 ␮s. Györke and Fill (112) used a
Ca2⫹ electrode to measure free Ca2⫹ concentrations before and after photolysis. Thus the properties of the fast
Ca2⫹ overshoot could not be measured. These kinetic
estimates predict that the fast Ca2⫹ overshoot was at least
three orders of magnitude faster than the observed adaptation phenomenon (112, 372).
B. Ca2ⴙ Overshoot Concern
The concern was that RyR2 adaptation might simply
be deactivation following the fast Ca2⫹ overshoot (154).
In other words, the Ca2⫹ activation sites will be rapidly
occupied during the overshoot. After the overshoot, occupancy of the site will fall and deactivate the channel.
The idea was that the RyR2 deactivation after the overshoot (lasting ⬍200 ␮s) generates the adaptation (␶ ⫽ ⬃1
s) phenomenon. If this were the case, then the deactivation of the RyR2 would need to be very slow. Is RyR2
Ca2⫹ deactivation so slow? The answer is no. The rate of
RyR2 channel deactivation in response to a fast Ca2⫹
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XVI. ADAPTATION DEBATE
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912
MICHAEL FILL AND JULIO A. COPELLO
C. Differing Interpretations
Several studies have now defined the kinetics of single RyR2 channel Ca2⫹ regulation (112, 113, 159, 264, 282,
283, 339, 340, 362). Although the data are generally consistent, how the different data sets have been interpreted
varied a great deal. Some investigators choose to interpret
their data in terms of adaptation (112, 339, 340), whereas
others choose to interpret their data in terms of classical
Ca2⫹-dependent inactivation (159, 264, 283). Several factors contributed to the different interpretations. The various studies used very different techniques and consequently applied very different types of Ca2⫹ stimuli. This
is important because even relatively small differences in
the characteristics of the Ca2⫹ stimulus can have a large
impact on single RyR2 channel function (81). For years,
the absence of adaptation following the mechanically inPhysiol Rev • VOL
FIG. 3. Markovian scheme of modal RyR2 channel gating. The channel recording is thought to reflect the fluctuation of the molecule between four highlighted sets of states, including high and low open
probability (Po) modes, to explain the bursting nature of the channel’s
gating. Below are four simulated single channel records generated using
this scheme. Each illustrates the predicted response to a fast steplike
free calcium change. The different colored regions of the recordings
correspond to the different colored sets of the gating scheme. [Adapted
from Zahradnı́ková and Zahradnı́k (361).]
duced Ca2⫹ stimuli was construed as evidence against the
adaptation phenomenon. A great deal of attention was
focused on understanding how the Ca2⫹ overshoot could
generate “adaptation.” Attention is now more appropriately focused on understanding how the different types of
Ca2⫹ stimuli may impact channel function. It is now clear
that the photolytic Ca2⫹ stimuli have a fast component
not present in the mechanical methods. Thus it is not
surprising that RyR channel behavior is different in the
different studies. The point is that the results of the flash
photolysis studies (112, 339, 340, 362) are not inherently
at odds with those reported in the mechanical solution
change studies (159, 160, 264, 282, 283), and there is no
reason a priori to favor one type of study over another.
Each study should be evaluated on its own merits, for
each represents a heroic effort and makes a substantive
contribution. In our opinion, the differences in the Ca2⫹
stimuli make the data more (not less) interesting. The fast
photolytic Ca2⫹ stimuli are particularly interesting because they may mimic the Ca2⫹ changes that activate RyR
channels in cells.
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reduction has been experimentally defined (264, 340, 362),
and it is quite fast (␶ values ⬃5 ms). Does the fast Ca2⫹
overshoot impact single RyR2 channel function? The answer is yes. Zahradnı́ková et al. (362) using improved
recording conditions showed that the fast Ca2⫹ overshoot, albeit slightly smaller than those applied by Györke
and Fill (112), can occasionally induce one or two brief
opening events. Application of Ca2⫹ stimuli like those
applied by Györke and Fill (112) (i.e., overshoot followed
by a sustained elevation), induce an entirely different
pattern of single RyR2 channel activity (including the
classical adaptation phenomenon). These data and more
recent single-channel modeling (86) suggest the impact of
the fast Ca2⫹ overshoot on single RyR2 channel function
may simply be to accelerate the initial opening transition.
Does the fast Ca2⫹ overshoot induce the adaptation
phenomenon? Few questions concerning single RyR
channel function have been debated more (86, 152, 288).
In principle, adaptation represents a slow transition of the
RyR channel from one state to another. There is circumstantial experimental evidence that suggests that this
slow transition is not simple Ca2⫹ deactivation following
the Ca2⫹ overshoot (154, 264, 340, 362). There are also
theoretical arguments that suggest that the slow transition is not induced by the fast Ca2⫹ overshoot. A specific
Markovian gating scheme, albeit still imperfect, has been
forwarded to describe RyR2 adaptation (see Fig. 3; Ref.
86). In this scheme, a fast Ca2⫹ overshoot is not required
to generate adaptation-like phenomenon. This scheme
suggests that RyR2 adaptation is due to a transient Ca2⫹dependent modal RyR2 gating shift (see sect. XVII and Fig.
3). There is, however, no experimental evidence directly
addressing the salient point (i.e., demonstration that the
same channel response is elicited by a Ca2⫹ step with or
without the initial overshoot). Thus the question above
has not been definitively answered yet.
RYANODINE RECEPTOR Ca2⫹ REGULATION
XVII. MODAL GATING
changes suddenly. Small fast Ca2⫹ elevations (from a low
Ca2⫹ level) upset the existing dynamic equilibrium momentarily. The result is a transient change in channel
activity until a new equilibrium is reached. Specifically, it
has been argued that single RyR2 channel activity momentarily rises to a Po level above that predicted by steadystate measurements before it spontaneously decays (86).
Channel activity decays as the channel reequilibrates between the gating modes at the new higher Ca2⫹ level (Fig.
4). Subsequent small fast Ca2⫹ elevations would induce
further transient modal gating shifts, inducing additional
transient episodes of high channel activity. This behavior
is reminiscent of the adaptation phenomenon. The magnitude of the transient modal gating shift should depend
on the speed and magnitude of the Ca2⫹ stimulus. A slow
Ca2⫹ stimulus would not induce a dramatic modal gating
shift because mode reequilibration is time dependent (i.e.,
modes will reequilibrate during the slow stimulus). Large
Ca2⫹ stimuli should be less effective because at high Ca2⫹
levels the channel will begin to frequent the inactivated
state. Consequently, a slow large Ca2⫹ stimulus would not
induce a substantial transient modal gating shift but may
instead induce Ca2⫹-dependent inactivation.
Is this consistent with the experimental record? Applications of fast Ca2⫹ elevations do indeed induce super
steady-state Po levels that are followed by a spontaneous
decay in channel activity (112, 159, 339). Also, repeated
episodes of transient channel activity in response to multiple incremental Ca2⫹ elevations have been reported
(112). Experiments have also defined the dependence of
channel activation rate on stimulus speed (159, 264, 283)
FIG. 4. Modal gating of the RyR2 channel. The Markovian scheme shown in Figure 3 was used here to simulate
the single-channel recordings shown. At the top, a series
of high Po bursts or low Po bursts are marked. Trains of
high or low Po bursts correspond to the high or low Po
gating modes of the RyR2 channel. At any steady-state
calcium concentration, the RyR2 channel will spontaneously shift between its different gating modes. A sample
spontaneous modal gating shift (at pCa 5) is shown in the
middle panel. In this case, the channel shifted from the
low Po mode to the high Po mode and back. Shifts in model
RyR2 gating can also be evoked by fast calcium concentration changes. A sample evoked modal gating shift is
shown on the bottom panel. A fast calcium concentration
change (pCa 7 to 6) was introduced at the arrow. The
channel immediately shifts into the high Po mode for a
period of time before shifting again into the low Po mode.
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Under steady-state conditions, single RyR2 channel
activity occurs in bursts. This means that channel openings tend to be clustered in time instead of being randomly distributed in time. The Po during a burst can be
either high or low (Fig. 4). The high Po and low Po bursts
do not occur randomly. Instead, the bursts tend to be
clustered as well. Trains of high Po bursts characterize a
high Po gating mode. Trains of low Po bursts characterize
a low Po gating mode. Extended periods of no openings
characterize a zero Po gating mode. Several different
groups have now reported the existence of modal RyR2
channel gating (8, 86, 261, 361).
How does modal RyR2 channel gating work? This is
still an open question. The identities, transitions, and
characteristics of the different RyR2 gating modes are
currently being defined. Preliminary interpretations suggest that there is a dynamic equilibrium between at least
three different gating modes under steady-state conditions (i.e., at a constant Ca2⫹ level). High, low, and zero Po
modes are clearly evident in the steady-state activity of
single RyR2 channels (8, 86, 261, 361). There also appears
to be clear spontaneous shifts between the different gating modes. Simulated single-channel data are presented in
Figure 4 to illustrate some of these points. It is thought
that the dynamic Ca2⫹-dependent equilibrium between
gating modes is what generates the classical bell-shaped
steady-state Ca2⫹ dependence of the RyR2 channel (54,
161, 353).
An interesting thing happens when the Ca2⫹ level
913
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MICHAEL FILL AND JULIO A. COPELLO
XVIII. CONCLUDING REMARKS
The advent of in vitro single RyR channel recording
has provided an abundance of information. It has revealed
key properties of the RyR channel’s permeation pathway
and how (and if) particular ligands alter channel function.
Despite some amazing advances, many fundamental questions about how the RyR channels are turned on and off
in vivo remain unanswered. For example, in vitro single
RyR2 channel recording has not definitively established
the identity of the mechanism(s) that terminate the SR
Ca2⫹ release process or revealed how movement of the
DHPR voltage sensor governs RyR1 channel activity.
Our understanding of single RyR channel regulation
is clearly incomplete. This impairs our understanding of
many intracellular calcium signaling phenomena. Longstanding RyR channel mysteries remain and are certain to
motivate research long into the future.
We thank the reviewers for their meticulous revision of the
manuscript and for providing us with very constructive comments. We also thank Dr. J. Ramos-Franco and M. Porta for
comments on the manuscript and A. Nani for technical help.
Research in the laboratory of M. Fill is supported by National Heart, Lung, and Blood Institute Grant HL-57832. J. A.
Copello is supported by American Heart Association Grant
0130142N.
Address for reprint requests and other correspondence: M.
Fill, Dept. of Physiology, Loyola University Chicago, 2160 South
First Ave., Maywood, IL 60153.
Physiol Rev • VOL
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and clear refractory behavior at very high Ca2⫹ levels
(264). Thus the modal gating theory qualitatively presented above has some experimental support. It is clear,
however, that additional experimental results are needed.
For example, current modal gating theory does not yet
consider the potential impact of certain physiologically
important ligands (e.g., luminal Ca2⫹, cytosolic Mg2⫹, and
ATP).
Is modal RyR channel gating physiological? Modal
gating may reproduce and reconcile certain single RyR
channel phenomena in bilayers, but its physiological relevance is questionable. In cells, the RyR channels generate intracellular Ca2⫹ signals that last less than ⬃25 ms
(e.g., sparks). Studies of “steady-state” RyR behavior in
bilayers report that intermodal gating transitions occur
over seconds. From these data, it appears unlikely that
modal RyR gating is a physiologically relevant phenomenon. Modal gating may simply be a biophysical ramification of RyR channel Ca2⫹ regulatory complexity. This
does not make it less important because understanding
the origin of modal gating may provide new insights into
RyR channel Ca2⫹ regulation.
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