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S L Hamilton

Publications and source records attributed to S L Hamilton.

At least 37 records · Page 2Linked to original sources
PubMed1997

Nitric oxide protects the skeletal muscle Ca2+ release channel from oxidation induced activation.

Reactive oxygen intermediates and nitric oxide modulate the contractile function of skeletal muscle fibers, possibly via direct interaction with the Ca2+ release channel. Oxidants produce disulfide bonds between subunits of the Ca2+ release channel tetramer, and this is accompanied by an increase in channel activity. The sulfhydryl alkylating agent N-ethylmaleimide has three distinct effects on Ca2+ release channel activity: first, channel activity is decreased (phase 1); then with continued exposure the activity is dramatically increased (phase 2); and finally, the channel is again inhibited (phase 3) (Aghdasi, B., Zhang, J. Z., Wu, Y., Reid, M. B., and Hamilton, S. L., (1997) J. Biol. Chem. 272, 3739-3749). Both H2O2 and nitric oxide (NO) block the phase 1 inhibitory effect of N-ethylmaleimide. NO donors, at concentrations that have no detectable effect on channel activity, block intersubunit cross-linking and prevent activation of the channel by the disulfide inducing agent, diamide. These findings support a model in which NO modulates the activity of the Ca2+ release channel by preventing oxidation of regulatory sulfhydryls. However, higher concentrations of NO donors activate the channel and produce intersubunit cross-links, supporting a bifunctional effect of NO on channel activity. Low NO concentrations prevent oxidation of the Ca2+ release channel whereas higher concentrations oxidize it.

Alkylation↗
PubMed1997

Functional interactions between cytoplasmic domains of the skeletal muscle Ca2+ release channel.

The skeletal muscle Ca2+ release channel (RYR1), which plays a critical role in excitation-contraction coupling, is a homotetramer with a subunit molecular mass of 565 kDa. Oxidation of the channel increases its activity and produces intersubunit cross-links within the RYR1 tetramer (Aghdasi, B., Zhang, J., Wu, Y., Reid, M. B., and Hamilton, S. L. (1997) J. Biol. Chem. 272, 3739-3748). Alkylation of hyperreactive sulfhydryls on RYR1 with N-ethylmaleimide (NEM) inhibits channel function and blocks the intersubunit cross-linking. We used calpain and tryptic cleavage, two-dimensional SDS-polyacrylamide gel electrophoresis, N-terminal sequencing, sequence-specific antibody Western blotting, and [14C]NEM labeling to identify the domains involved in these effects. Our data are consistent with a model in which 1) diamide, an oxidizing agent, simultaneously produces an intermolecular cross-link between adjacent subunits within the RYR1 tetramer and an intramolecular cross-link within a single subunit; 2) all of the cysteines involved in both cross-links are in either the region between amino acids approximately 2100 and 2843 or the region between amino acids 2844 and 4685; 3) oxidation exposes a new calpain cleavage site in the central domain of the RYR1 (in the region around amino acid 2100); 4) sulfhydryls that react most rapidly with NEM are located in the N-terminal domain (between amino acids 426 and 1396); 5) alkylation of the N-terminal cysteines completely inhibits the formation of both inter- and intrasubunit cross-links. In summary, we present evidence for interactions between the N-terminal region and the putatively cytoplasmic central domains of RYR1 that appear to influence subunit-subunit interactions and channel activity.

Animals↗
PubMed1997

Factors influencing [3H]ryanodine binding to the skeletal muscle Ca2+ release channel.

Optimal [3H]ryanodine binding to skeletal muscle sarcoplasmic reticulum membranes is dependent on a number of factors such as Ca2+ concentration, ionic strength, and the presence of modulators of the Ca2+ release channel. The rate of association of [3H]-ryanodine with its binding site is slower than a diffusion limited process, and often the binding reaches a peak value which is followed by a slow decline. This phenomenon makes it extremely difficult to determine kinetic constants for [3H]ryanodine binding. The inclusion of bovine serum albumin (BSA) or the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) in the incubation buffer prevents the decrease in [3H]ryanodine binding observed in association studies. BSA or Chaps slows this decline in binding partially by preventing a conversion to a more rapidly dissociating component. Pretreatment of the membranes with Chaps does not prevent the decrease in [3H]ryanodine binding, suggesting that Chaps is not exerting its effect by extracting a lipid or peripheral membrane protein. The decrease in affinity observed in the absence of BSA and Chaps appears to require the occupation of the high-affinity ryanodine binding site. Incubation for extended times in the absence of [3H]ryanodine prior to the initiation of the association produced similar curves to those obtained without preincubation. These combined results suggest that Chaps and BSA stabilize the ryanodine-modified Ca2+ release channel by preventing an alteration in the ryanodine binding site which leads to decreased affinity, thus allowing for a more quantitative interpretation of binding data.

Animals↗
PubMed1997

Multiple classes of sulfhydryls modulate the skeletal muscle Ca2+ release channel.

Two sulfhydryl reagents, N-ethylmaleimide (NEM), an alkylating agent, and diamide, an oxidizing agent, were examined for effects on the skeletal muscle Ca2+ release channel. NEM incubated with the channel for increasing periods of time displays three distinct phases in its functional effects on the channel reconstituted into planar lipid bilayers; first it inhibits, then it activates, and finally it again inhibits channel activity. NEM also shows a three-phase effect on the binding of [3H]ryanodine by first decreasing binding (phase 1), followed by a recovery of the binding (phase 2), and then a final phase of inhibition (phase 3). In contrast, diamide 1) activates the channel, 2) enhances [3H]ryanodine binding, 3) cross-links subunits within the Ca2+ release channel tetramer, and 4) protects against phase 1 inhibition by NEM. All diamide effects can be reversed by the reducing agent, dithiothreitol. Diamide induces intersubunit dimer formation of both the full-length 565-kDa subunit of the channel and the 400-kDa generated by endogenous calpain digestion, suggesting that the cross-link does not involve sulfhydryls within the N-terminal 170-kDa fragment of the protein. NEM under phase 1 conditions blocks the formation of the intersubunit cross-links by diamide. In addition, single channels activated by diamide are further activated by the addition of NEM. Diamide either cross-links phase 1 sulfhydryls or causes a conformational change in the Ca2+ release channel which leads to inaccessibility of phase 1 sulfhydryls to NEM alkylation. The data presented here lay the groundwork for mapping the location of one of the sites of subunit-subunit contact in the Ca2+ release channel tetramer and for identifying the functionally important sulfhydryls of this protein.

Animals↗
PubMed1997

Modulation of skeletal muscle Ca2(+)-release channel activity by sphingosine.

The effect of D-erythro-C18-sphingosine (sphingosine) and related compounds on the Ca(2+)-release channel (ryanodine binding protein) was examined on rabbit skeletal muscle membranes, on the purified ryanodine binding protein, and on the channel reconstituted into planar lipid bilayers. Sphingosine inhibited [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes in a dose-dependent manner similar to published results (R. A. Sabbadini, R. Betto, A. Teresi, G. Fachechi-Cassano, and G. Salviati. J. Biol. Chem. 267: 15475-15484, 1992). The sphingolipid also inhibited [3H]ryanodine binding to the purified ryanodine binding protein. Our results demonstrate that the inhibition of [3H]ryanodine binding by sphingosine is due to an increased rate of dissociation of bound [3H]ryanodine from SR membranes and a decreased rate of association of [3H]ryanodine to the high-affinity site. Unlike other modulators of the Ca(2+)-release channel, sphingosine can remove bound [3H]ryanodine from the high-affinity site within minutes. Sphingosine increased the rate of dissociation of [3H]ryanodine bound to a solubilized proteolytic fragment derived from the carboxy terminus of the ryanodine binding protein (cleavage at Arg4475). Sphingosine also inhibited the activity of the Ca(2+)-release channel incorporated into planar lipid bilayers. Taken together, the data provide evidence for a direct effect of sphingosine on the Ca(2+)-release channel. Sphingosine is a noncompetitive inhibitor at the high-affinity ryanodine binding site, and it interacts with a site between Arg4475 and the carboxy terminus of the Ca(2+)-release channel.

Animals↗
PubMed1997

A carboxy-terminal peptide of the alpha 1-subunit of the dihydropyridine receptor inhibits Ca(2+)-release channels.

Excitation-contraction coupling in skeletal muscle is thought to involve a physical interaction between the alpha 1-subunit of the dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) Ca(2+)-release channel (also known as the ryanodine receptor). Considerable evidence has accumulated to suggest that the cytoplasmic loop between domains II and III of the DHPR alpha 1-subunit is at least partially responsible for this interaction. Other parts of this subunit or other subunits may, however, contribute to the functional and/or structural coupling between these two proteins. A synthetic peptide corresponding to a conserved sequence located between amino acids 1487 and 1506 in the carboxy terminus of the alpha 1-subunit inhibits both [3H]ryanodine binding to skeletal and cardiac SR membranes and the activity of skeletal SR Ca(2+)-release channels reconstituted into planar lipid bilayers. A second, multiantigenic peptide synthesized to correspond to the same sequence inhibits both binding and channel activity at lower concentrations than the linear peptide. These peptides slow the rate at which [3H]ryanodine binds to its high-affinity binding site and decrease the rate at which [3H]ryanodine dissociates from this site. A third polypeptide synthesized in Escherichia coli and corresponding to amino acids 1381-1627 and encompassing the above sequence has similar effects. This portion of the alpha 1-subunit of the transverse tubule DHPR is therefore a candidate for contributing to the interaction of this protein with the Ca(2+)-release channel.

Animals↗
PubMed1996

Interaction between ryanodine and neomycin binding sites on Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

Neomycin is a potent inhibitor of skeletal muscle sarcoplasmic reticulum (SR) calcium release. To elucidate the mechanism of inhibition, the effects of neomycin on the binding of [3H]ryanodine to the Ca2+ release channel and on its channel activity when reconstituted into planar lipid bilayer were examined. Equilibrium binding of [3H]ryanodine was partially inhibited by neomycin. Inhibition was incomplete at high neomycin concentrations, indicating noncompetitive inhibition rather than direct competitive inhibition. Neomycin and [3H]ryanodine can bind to the channel simultaneously and, if [3H]ryanodine is bound first, the addition of neomycin will slow the dissociation of [3H]ryanodine from the high affinity site. Neomycin also slows the association of [3H]ryanodine with the high affinity binding site. The neomycin binding site, therefore, appears to be distinct from the ryanodine binding site. Dissociation of [3H]ryanodine from trypsin-treated membranes or from a solubilized 14 S complex is also slowed by neomycin. This complex is composed of polypeptides derived from the carboxyl terminus of the Ca2+ release channel after Arg-4475 (Callaway, C., Seryshev, A., Wang, J. P., Slavik, K., Needleman, D. H., Cantu, C., Wu, Y., Jayaraman, T., Marks, A. R., and Hamilton, S. L. (1994) J. Biol. Chem. 269, 15876-15884). The proteolytic 14 S complex isolated with ryanodine bound produces a channel upon reconstitution into planar lipid bilayers, and its activity is inhibited by neomycin. Our data are consistent with a model in which the ryanodine binding sites, the neomycin binding sites, and the channel-forming portion of the Ca2+ release channel are located between Arg-4475 and the carboxyl terminus.

Animals↗
PubMed1996

Two structural configurations of the skeletal muscle calcium release channel.

Here we present the determination of the three-dimensional structure of the skeletal muscle Ca2+-release channel in an open state using electron cryomicroscopy and angular reconstitution. In contrast to our reconstruction of the channel in its closed state, the density map of the channel driven towards its open state, by the presence of Ca2+ and ryanodine, features a central opening in the transmembrane region-the likely passageway for Ca2+ ions from the sarcoplasmic reticulum to the cytosol. The opening of the channel is associated with a 4 degree rotation of its transmembrane region with respect to its cytoplasmic region, and with significant mass translocations within the entire cytoplasmic region of the channel tetramer.

Animals↗
PubMed1995

Effect of ryanodine on sarcoplasmic reticulum Ca2+ accumulation in nonfailing and failing human myocardium.

BACKGROUND: The purpose of this study was to determine whether abnormal Ca2+ release through ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum might contribute to the abnormal [Ca2+]i homeostasis that has been described in failing human myocardium. METHODS AND RESULTS: Occupancy of low-affinity ryanodine binding sites on ryanodine-sensitive Ca2+ channels stimulates oxalate-supported, ATP-dependent Ca2+ accumulation in sarcoplasmic reticulum-derived microsomes by inhibiting concurrent Ca2+ efflux through these channels. We examined the effects of 0.5 mmol/L ryanodine on 45Ca2+ accumulation in microsomes prepared from nonfailing (n = 8) and failing (n = 10) human left ventricular myocardium. In the absence of ryanodine, 45Ca2+ accumulation reached similar levels in microsomes from nonfailing and failing hearts. Incubation with 0.5 mmol/L ryanodine caused a 52.2 +/- 6.5% increase in peak 45Ca2+ accumulation in microsomes from nonfailing hearts and a 24.3 +/- 4.1% increase in microsomes from failing hearts. The density of high-affinity ryanodine binding sites and the inhibition of [3H]ryanodine dissociation from these sites by 0.1 mmol/L ryanodine were similar in microsomes from nonfailing and failing hearts. CONCLUSIONS: These results, which demonstrate a diminished stimulation of Ca2+ accumulation by ryanodine in sarcoplasmic reticulum-derived microsomes from failing human myocardium that could be explained by an uncoupling of the occupancy of low-affinity ryanodine binding sites from the reduction in the open probability of these channels or by concurrent Ca2+ efflux through a ryanodine-insensitive mechanism, are evidence that increased efflux of Ca2+ from the sarcoplasmic reticulum may contribute to the abnormal [Ca2+]i homeostasis described in failing human myocardium.

Adult↗
PubMed1995

Identification of calcium release-triggering and blocking regions of the II-III loop of the skeletal muscle dihydropyridine receptor.

In an attempt to identify and characterize functional domains of the rabbit skeletal muscle dihydropyridine receptor alpha 1 subunit II-III loop, we synthesized several peptides corresponding to different regions of the loop: peptides A, B, C, C1, C2, D (cf. Fig. 1). Peptide A (Thr671-Leu690) activated [3H]ryanodine binding to, and induced Ca2+ release from, rabbit skeletal muscle triads, but none of the other peptides had such effects. Peptide A-induced Ca2+ release and activation of ryanodine binding were partially suppressed by an equimolar concentration of peptide C (Glu724-Pro760) but were not affected by the other peptides. These results suggest that the short stretch in the II-III loop, Thr671-Leu690, is responsible for triggering SR Ca2+ release, while the other region, Glu724-Pro760, functions as a blocker of the release trigger. A hypothesis is proposed to account for how these subdomains interact with the sarcoplasmic reticulum Ca2+ release channel protein during excitation-contraction coupling.

Amino Acid Sequence↗
PubMed1995

Electron cryomicroscopy and angular reconstitution used to visualize the skeletal muscle calcium release channel.

We exploit the random orientations of ice-embedded molecules imaged in an electron cryomicroscope to determine the three-dimensional structure of the Ca(2+)-release channel from the sarcoplasmic reticulum (SR) in its closed state, without tilting the specimen holder. Our new reconstruction approach includes an exhaustive search of all different characteristic projection images in the micrographs and the assignment of Euler angle orientations to these views. The 30 A map implied reveals a structure in which the transmembrane region exhibits no apparent opening on the SR lumen side. The extended cytoplasmic region has a hollow appearance and consists, in each monomer, of a clamp-shaped and a handle-shaped domain.

Animals↗
PubMed1994

Localization of the high and low affinity [3H]ryanodine binding sites on the skeletal muscle Ca2+ release channel.

The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant alkaloid ryanodine and there are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino acids. The ability of sarcoplasmic reticulum membranes to bind [3H]ryanodine to the high affinity site is lost upon proteolysis with trypsin. [3H]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [3H]ryanodine and then 100 microM ryanodine is added to occupy the low affinity sites, almost all of [3H]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubilized Ca2+ release channel containing bound [3H]ryanodine to undergo four discrete shifts in sedimentation (30 S-->28 S-->26 S-->19 S-->14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56-, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are recognized by an antibody to the last 9 amino acids at the carboxyl terminus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37-kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2+ release channel fragments. Both high and low affinity ryanodine binding sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.

Amino Acid Sequence↗
PubMed1993

Relationship of low affinity [3]ryanodine binding sites to high affinity sites on the skeletal muscle Ca2+ release channel.

Both high and low affinity binding sites for [3H]ryanodine exist in sarcoplasmic reticulum membranes derived from rabbit skeletal muscle. Negatively cooperative binding of [3H]ryanodine at one of four initially identical sites cannot account for some of the kinetic features of the binding to high and low affinity sites. The presence of excess unlabeled ryanodine greatly slows the rate at which [3H]ryanodine bound at the high affinity site dissociates. An examination of the rate of dissociation of [3H]ryanodine bound at increasing [3H]ryanodine concentrations reveals the existence of a second site, occupied only at high ligand concentrations. The occupation of this site correlates well with the conversion of the high affinity site from a site with a dissociation rate constant of approximately 0.0025 min-1 to one with a dissociation rate constant of less than 0.00025 min-1. The low affinity site itself has a dissociation rate constant of 0.013 min-1 and dissociation from this site is unaffected by the presence of 100 microM unlabeled ryanodine. These data suggest that the two binding sites are different but are either allosterically or sterically coupled. Association experiments support this interpretation. Low affinity binding sites for [3H]ryanodine exist in transverse tubule (t-tubule) as well as sarcoplasmic reticulum membranes. High concentrations of both ryanodine and ruthenium red inhibit the binding of [3H]PN200-110 to the dihydropyridine-binding protein in t-tubule membranes. Whether the low affinity site in t-tubule membranes is related to that found in sarcoplasmic reticulum membranes is not yet known.

Animals↗
PubMed1992

[3H]ryanodine as a probe of changes in the functional state of the Ca(2+)-release channel in malignant hyperthermia.

The defect in malignant hyperthermia (MH) alters the binding of [3H]ryanodine to the Ca(2+)-release channel by increasing its apparent affinity for the binding site. In sarcoplasmic reticulum (SR) membranes from both normal and mutant pigs the apparent Kd is dependent on a number of parameters. Adenosine 5'-(beta,gamma-methylene)triphosphate, ionic strength, and Ca2+ each increase the apparent affinity of the binding site for [3H]ryanodine. Equilibrium and kinetic evaluation of the binding of [3H]ryanodine to these membranes demonstrates that the MH defect in pigs increases the apparent affinity of the membranes for [3H]ryanodine by increasing the amount of high affinity relative to low affinity binding sites. Both the association and dissociation of [3H]ryanodine with all three types of membranes (normal, heterozygous MH, homozygous MH) are characterized by two or more components, with the relative ratios of these components altered by the MH defect. These findings suggest that the observed Kd is the weighted average of the binding of ryanodine to two or more interconvertible states of the channel. Dilution of [3H]ryanodine bound to normal membranes at high Ca2+ into low Ca2+ solutions enhances the rate of dissociation. This conversion occurs to a much lesser extent with MH membranes, suggesting that the MH defect may alter the rate at which the high affinity form of the protein converts to the low affinity form.

Animals↗
PubMed1991

Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle.

The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs.

Blotting, Western↗
PubMed1991

Regulation of the sarcoplasmic reticulum Ca(2+)-release channel requires intact annexin VI.

Annexin VI has eight highly conserved repeated domains; all other annexins have four. Díaz-Muñoz et al. (J Biol Chem 265:15894, 1990) reported that annexin VI alters the gating properties of the ryanodine-sensitive Ca(2+)-release channel isolated from sarcoplasmic reticulum. The investigate the domain structure of rat annexin VI (67 kDa calcimedin) required for this channel regulation, various proteolytic digestions were performed. In each case, protease-resistant core polypeptides were produced. Annexin VI was digested with V8 protease and two core polypeptides were purified by Ca(2+)-dependent phospholipid binding followed by HPLC. The purified fragments were shown to be derived from the N- and C-terminal halves of annexin VI, and demonstrated differential immunoreactivity with monoclonal antibodies to rat annexin VI. While both core polypeptides retained their ability to bind phospholipids in a Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-release channel as did intact annexin VI.

Amino Acid Sequence↗
PubMed1990

Modulation of Ca2+ release channel activity from sarcoplasmic reticulum by annexin VI (67-kDa calcimedin).

The effect of annexin VI (67-kDa calcimedin) on the activity of the Ca2+ release channel was studied using heavy sarcoplasmic reticulum membranes reconstituted into planar bilayers. Annexin VI, in a range of 5-40 nM, modified the gating behavior of the Ca2+ release channel by increasing the probability of opening by 2.7-fold and the mean open time by 82-fold relative to controls. Annexin VI caused no change in the slope conductance of the channel. The modulatory effect of annexin VI on the activity of Ca2+ release channels was Ca2+ dependent, and the annexin VI-modified channel was sensitive to both ruthenium red and ryanodine. The effect of annexin VI was observed when this protein was added specifically to the trans chamber, which corresponds to the luminal side of sarcoplasmic reticulum as determined by the ATP activation of the channel. In addition, differential extraction studies demonstrated that some annexin VI is localized within the lumen of the isolated heavy sarcoplasmic reticulum vesicles prepared by several different procedures. Annexin VI did not modify, from either the cis or trans chambers, the activity of K+ or Cl- channels from sarcoplasmic reticulum or the dihydropyridine sensitive Ca2+ channel from transverse tubules. In addition, the 38-kDa core proteolytic fragments of annexin VI had no effect on the Ca2+ release channel activity. Annexin VI is therefore a candidate for a physiological modulator of the Ca2+ release channel and as such, may play an important role in the excitation-contraction coupling.

Animals↗