I. INTRODUCTION

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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

	II. HISTORY

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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⁺ 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 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

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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 RyR3-knockout mice appears to be impaired (357). The implications 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 putative 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 structure-function 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 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 molecules (~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

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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.

View larger version (58K): [in this window] [in a new window]   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 DHPR-RyR1 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.

View larger version (64K): [in this window] [in a new window]   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.

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.

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).

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 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 DHPR-RyR1 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).

	V. STUDY OF INTRACELLULAR CALCIUM RELEASE

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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 channel or more likely the concerted opening of a small number of RyR channels.

	VI. STUDY OF SINGLE RYANODINE RECEPTOR CHANNELS

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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 (P_o) 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.

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 Tris-HEPES 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).

	VII. PERMEATION PROPERTIES

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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 P_o 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 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-K_Ca 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 (P_Ca/P_K ~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 (P_Ca/P_K >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 high-conductance 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 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.

	VIII. ACTION OF RYANODINE

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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 induces 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 µM) are reported to induce the classical ryanodine action described above (34, 259, 294). High doses of ryanodine (~100 µM) 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 (K_D <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 (K_D >1 µM) 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

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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 red). Some of these modulatory factors are discussed below.

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 µM) 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.

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 regulation). Distinguishing between these possibilities is the challenge.

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 µM 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).

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.

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.

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 P_o 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.

	X. REGULATION BY CLOSELY ASSOCIATED PROTEINS

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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.

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 DHPR-RyR1 interaction (117, 269). A potential role of CaM in modulation of the RyR2 channel during E-C coupling is not yet clear.

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, 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).

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 single-channel function neighboring RyRs (188). The suggestion is that this synchronization permits several neighboring RyR channels to open and close simultaneously. It is not 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 FKBP12.6-RyR2 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.

Another protein that interacts with and regulates the function of single RyR channels is the DHPR (323). As 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 DHPR-RyR1 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.

	XI. CALCIUM REGULATIO