Skeletal Muscle Sarcoplasmic Reticulum Membranes Research Articles

No voltage change at skeletal muscle SR membrane during Ca2+ release-just Mermaids on acid.

Calcium ions control multiple physiological functions by binding to extracellular and intracellular targets. One of the best-studied Ca2+-dependent functions is contraction of smooth and striated muscle tissue, which results from Ca2+ ligation to calmodulin and troponin C, respectively. Ca2+ signaling typically involves flux of the ion across membranes via specifically gated channel proteins. Because calcium ions are charged, they possess the ability to generate changes in the respective transmembrane voltage. Ca2+-dependent voltage alterations of the surface membrane are easily measured using microelectrodes. A well-known example is the characteristic plateau phase of the action potential in cardiac ventricular cells that results from the opening of voltage-gated L-type Ca2+ channels. Ca2+ ions are also released from intracellular storage compartments in many cells, but these membranes are not accessible to direct voltage recording with microelectrodes. In muscle, for example, release of Ca2+ from the sarcoplasmic reticulum (SR) to the myoplasm constitutes a flux that is considerably larger than the entry flux from the extracellular space. Whether this flux is accompanied by a voltage change across the SR membrane is an obvious question of mechanistic importance and has been the subject of many investigations. Because the tiny spaces enclosed by the SR membrane are inaccessible to microelectrodes, alternative methods have to be applied. In a study by Sanchez et al. (2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812035) in this issue, modern confocal light microscopy and genetically encoded voltage probes targeted to the SR were applied in a new approach to search for changes in the membrane potential of the SR during Ca2+ release.

Discovery of Tricyclic Clerodane Diterpenes as Sarco/Endoplasmic Reticulum Ca(2+)-ATPase Inhibitors and Structure-Activity Relationships.

Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 μM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented.

S100A1: a physiological modulator of RYR1, Ca2+ release, and contractility in skeletal muscle. Focus on “S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle”

SKELETAL MUSCLES adjust their contractile output over an exceptionally wide dynamic range, as needed to produce movements ranging from sitting and standing, to athletics and dance. At the single fiber level, the force generated by skeletal muscles is determined, ultimately, by the formation of cycling cross bridges between actin and myosin. This interaction requires Ca 2 and is governed by the simple relationship: force is proportional to intracellular ionized Ca 2 . Cross-bridge formation is inhibited in resting muscle when Ca 2 is sequestered within the sarcoplasmic reticulum (SR); it is activated when stored Ca 2 is released into the cytosol, allowing actin-myosin interactions to proceed. Contraction stops as Ca 2 is actively resequestered by the Ca 2 -ATPase of the SR (SERCA1). Thus Ca 2 is the central messenger in muscle activation, and the amplitude and time course of the intracellular Ca 2 change is the major determinant of contractile output. The key mechanisms that activate Ca 2 release from the SR are now well defined. This sequence, termed excitation-contraction coupling, begins when an excitatory input from the motor nerve triggers a propagating action potential on the muscle membrane. The action potential rapidly depolarizes the entire sarcolemma, including transverse tubules, which invaginate from the surface membrane and form specialized junctions (triads) with the SR along much of their length. The triad junction serves as a platform for assembling sarcolemmal calcium

Over-expression of Microspan, a novel component of the sarcoplasmic reticulum, causes severe muscle pathology with triad abnormalities

Sarcospan (SSPN) is a core component of the dystrophin-glycoprotein complex (DGC). Multiple SSPN transcripts are ubiquitously expressed and SSPN splicing is disrupted in many lung tumors, suggesting the importance of SSPN-related mRNAs. We describe the isolation of an alternatively spliced isoform of SSPN, which we designate 'microspan' based on its small size relative to SSPN. Microspan has two transmembrane domains and a novel C-terminus. We demonstrate that microspan is not an integral component of the DGC and is not perturbed by the loss of dystrophin. Microspan protein is detected at the sarcoplasmic reticulum (SR) using indirect immunofluorescence and immunoelectron microscopy. Furthermore, microspan purifies with skeletal muscle SR membranes and not transverse tubules. Mice engineered to over-express microspan display severe kyphosis and die at approximately 8 weeks of age. Levels of ryanodine receptor, dihydropyridine receptor, and SERCA-1 are greatly reduced in microspan transgenic muscle. Furthermore, electron microscopy reveals that microspan over-expression causes a dramatic perturbation in triad structure. Our findings suggest that microspan is an important component of the SR and may contribute to excitation-contraction coupling.

Malignant hyperthermia: a pharmacogenetic disease of Ca++ regulating proteins.

Malignant hyperthermia (MH) is a pharmacogenetic, life-threatening hypermetabolic syndrome in genetically predisposed individuals exposed to certain anesthetic agents. Discovered by Denborough and Lovell [1] in 1960, MH was associated with high mortality and morbidity as the cause was unknown and an effective treatment was unavailable. There is no classic clinical presentation of the syndrome, and the onset and signs of MH are dependent upon known and unknown environmental and genetic factors. Initial theories involved central temperature regulation defects or uncoupling of oxidative phosphorylation in mitochondria [2], but later investigations targeted skeletal muscle as the affected organ. Subsequently freshly biopsied skeletal muscle was used for in vitro pharmacologic contracture testing to discriminate between normal and MH-affected muscle and remains the "gold standard" for MH diagnosis. Spontaneous, genetic models for MH were discovered in pigs and dogs and substantial knowledge about MH was gained from these valuable resources. The abnormal contracture response of MH skeletal muscle evoked a focus on calcium regulation, and abnormalities in calcium release (as opposed to calcium sequestration) mechanisms were discovered. About this same time the major calcium release channel in the skeletal muscle sarcoplasmic reticulum membrane was purified and named the ryanodine receptor [3]. Although the ryanodine receptor represents one of the largest functional proteins, the enormous gene encoding the 5021 amino acids comprising the ryanodine receptor subunit was eventually cloned [4,5]. Patient and dedicated work on the ryanodine receptor gene has found linkage to MH in the pig [6], dog [7], and among several different mutations and MH in unrelated human families [8,9]. Expression of these mutations in HEK cells has resulted in abnormal calcium release [10,11], supporting but not proving a causal basis for MH. In this review each of the areas mentioned above is discussed in detail revealing a wonderful success story that changed the anesthesiologist's "worst nightmare" from a syndrome with high mortality and morbidity to a reasonably well managed disease today. This success story includes unraveling the molecular basis for the disease and brings its pathoetiologic and diagnostic aspects toward molecular genetic resolution.

Inhibition of Ca 2+ release channel (ryanodine receptor) activity by sphingolipid bases: mechanism of action

Sphingosine inhibits the activity of the skeletal muscle Ca 2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [ 3H]ryanodine binding (Needleman et al., Am. J. Physiol. 272, C1465-1474, 1997). To determine the contribution of other sphingolipids to the regulation of ryanodine receptor activity, several sphingolipid bases were assessed for their ability to alter [ 3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes and to modulate the activity of the Ca 2+ release channel. Three lipids, N, N-dimethylsphingosine, dihydrosphingosine, and phytosphingosine, inhibited [ 3H]ryanodine binding to both skeletal and cardiac SR membranes. However, the potency of these three lipids and sphingosine was lower in rabbit cardiac membranes when compared to rabbit skeletal muscle membranes and when compared to sphingosine. Like sphingosine, the lipids inhibited [ 3H]ryanodine binding by greatly increasing the rate of dissociation of bound [ 3H]ryanodine from SR membranes, indicating that these three sphingolipid bases were noncompetitive inhibitors of [ 3H]ryanodine binding. These bases also decreased the activity of the Ca 2+ release channel incorporated into planar lipid bilayers by stabilizing a long closed state. Sphingosine-1-PO 4 and C 6 to C 18 ceramides of sphingosine had no significant effect on [ 3H]ryanodine binding to cardiac or skeletal muscle SR membranes. Saturation of the double bond at positions 4–5 decreased the ability of the sphingolipid bases to inhibit [ 3H]ryanodine binding 2–3 fold compared to sphingosine. In summary, our data indicate that other endogenous sphingolipid bases are capable of modulating the activity of the Ca 2+ release channel and as a class possess a common mechanism of inhibition.

3H]Azidodantrolene: synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum.

Dantrolene sodium is a medically important hydantoin derivative that interferes with release of Ca2+ from intracellular stores of skeletal muscle by an unknown mechanism. Identification of the molecular target of dantrolene would greatly aid in understanding both the mechanism of action of the drug and the dynamics of intracellular Ca2+ release in muscle. [3H]Azidodantrolene was designed and synthesized as a photoaffinity analogue in order to identify a putative dantrolene receptor in skeletal muscle. Introduction of 1 mole-atom of tritium into aldehyde 5b was required during radioligand synthesis in order to ensure high enough specific activity for detection of photo-cross-linked proteins by fluorographic methods. This was accomplished by reduction of ester 3 with custom synthesized, 100% tritium-labeled lithium triethylborotritide, followed by oxidation to 5b by manganese(IV) oxide. Compound 6b was demonstrated to be >/=95% tritium-labeled at the imine position by NMR spectroscopy, and the specific radioactivity of [3H]azidodantrolene sodium was empirically determined by HPLC and liquid scintillation counting to be 24.4 Ci/mmol, approximately 85% of theoretical maximum. [3H]Azidodantrolene was found to be pharmacologically active in ligand-receptor binding studies with skeletal muscle sarcoplasmic reticulum membranes. Photo-cross-linking experiments analyzed by SDS-PAGE and tritium fluorography have identified a approximately 160-kDa specifically labeled protein as the putative, intracellular, skeletal muscle dantrolene receptor. This photolabeled protein comigrates with a protein in Western blots immunologically cross-reactive to a polyclonal anti-rabbit skeletal muscle ryanodine receptor antibody. Thus, the putative dantrolene receptor may be related to the skeletal muscle ryanodine receptor.

Pharmacological distinction between dantrolene and ryanodine binding sites: evidence from normal and malignant hyperthermia-susceptible porcine skeletal muscle.

Dantrolene inhibits and ryanodine stimulates calcium release from skeletal-muscle sarcoplasmic reticulum (SR), the former by an unknown mechanism, and the latter by activating the ryanodine receptor (RyR), the primary Ca2+-release channel of SR. Dantrolene is used to treat malignant hyperthermia (MH), a genetic predisposition to excessive intracellular Ca2+ release upon exposure to volatile anaesthetics. Porcine MH results from a point mutation in the SR RyR that alters the open probability of the channel, and is reflected in altered [3H]ryanodine binding parameters. Specific binding sites for [3H]dantrolene and [3H]ryanodine co-distribute on SR that has been isolated by discontinuous sucrose gradient centrifugation. If the two drug-binding sites are functionally linked, [3H]dantrolene binding might be affected both by pharmacological and by genetic modulators of the functional state of the RyR. Accordingly, we compared the characteristics of [3H]dantrolene binding to porcine malignant-hyperthermia-susceptible and normal-skeletal-muscle SR, and examined the effects of RyR modulators on [3H]dantrolene binding to these membranes. Additionally, the feasibility of separating the SR binding sites for [3H]dantrolene and [3H]ryanodine was investigated. No significant differences in [3H]dantrolene binding characteristics to SR membranes from the two muscle types were detected, and the Bmax ratio for [3H]dantrolene/[3H]ryanodine was 1.4(+/-0.1):1 in both muscle types. [3H]Dantrolene binding is unaffected by the RyR modulators caffeine, ryanodine, Ruthenium Red and calmodulin, and neither dantrolene nor azumolene have any effect on [3H]ryanodine binding. Additionally, distinct peaks of [3H]dantrolene and [3H]ryanodine binding are detected in SR membranes fractionated by linear sucrose centrifugation, although no differences in protein patterns are detected by SDS/PAGE or Western-blot analysis. We suggest that the binding sites for these two drugs are pharmacologically distinct, and may exist on separate molecules.

Identification of oxidation-sensitive peptides within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+-ATPase.

We have examined the oxidative sensitivity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) membranes, exposing isolated SR membranes to the thermolabile water soluble free radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Incubation with up to 702 microM AAPH-derived radicals results in a concentration- and time-dependent inhibition of calcium-dependent ATPase activity correlating with the loss of monomeric Ca2+-ATPase polypeptides, and the concomitant appearance of higher molecular weight species. However, no oxidant-induced protein fragmentation is detected. The observed formation of oxidant-induced bityrosine accounts for the intermolecular Ca2+-ATPase cross-links, as well as intramolecular cross-links. The oxidation of sulfhydryl groups to disulfides as another possible source of intermolecular cross-links has been ruled out after examination of SDS -PAGE performed under both reducing and non-reducing conditions. Exposure of the SR membranes to AAPH-derived radical species results in a small degree of lipid peroxidation that is not correlated with enzyme inactivation, suggesting that modification of membrane-spanning peptides is not related to enzyme inactivation. Six cytoplasmic peptides have been identified that are modified by exposure to AAPH or, alternatively, to hydrogen peroxide, suggesting that these regions of the Ca2+-ATPase are generally sensitive to oxidants. These oxidized peptides were identified after separation by reversed-phase HPLC followed by N-terminal sequencing and amino acid analysis as corresponding to the following sequences of the Ca2+-ATPase: (i) Glu121 to Lys128, (ii) His190 to Lys218, (iii) Asn330 to Lys352, (iv) Gly432 to Lys436, (v) Glu551 to Arg604, and (vi) Glu657 to Arg671. The Glu551 to Arg604 peptide, located within the nucleotide binding domain, was found to participate in the formation of intermolecular bityrosine cross-links with the identical Glu551 to Arg604 peptide from a neighboring Ca2+-ATPase polypeptide chain.

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-propane-sulfonate (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.

Self-association accompanies inhibition of Ca-ATPase by thapsigargin

Recent studies have demonstrated a relationship between the activity of the Ca-ATPase of sarcoplasmic reticulum and its state of self-association. In the present study, the effects of thapsigargin (TG), a toxin that specifically inhibits the Ca-ATPase of rabbit skeletal muscle sarcoplasmic reticulum membrane, were studied by detecting the time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase that had been labeled with the phosphorescent probe erythrosin-isothiocyanate (ErITC). Anisotropy decays were fit to a function that consisted of three exponential decays plus a constant background, as well as to a function describing explicitly the uniaxial rotation of proteins in a membrane. In the absence of TG, the anisotropy was best-fit by a model representing the rotation of three populations, corresponding to different-sized oligomeric species in the membrane. The addition of stoichiometric amounts of TG to the Ca-ATPase promptly decreased the overall apparent rate of decay, indicating decreased rotational mobility. A detailed analysis showed that the principal change was not in the rates of rotation but rather in the population distribution of the Ca-ATPase molecules among the different-sized oligomers. TG decreased the proportion of small oligomers and increased the proportion of large ones. Preincubation of the ErITC-SR in 1 mM Ca2+, which stabilizes the E1 conformation relative to E2, was found to protect partially against the changes in the TPA associated with the presence of the inhibitor. These results are consistent with the hypothesis that TG inhibits the Ca-ATPase by stabilizing it in an E2-like conformation, which promotes the formation of larger aggregates of the enzyme. When combined with the effects of other inhibitors on the Ca-ATPase, these results support a general model for the coupling of enzyme conformation and self-association in this system.

Reversible inactivation of the sarcoplasmic reticulum Ca(2+)-ATPase coupled to rearrangement of cytoplasmic protein domains as revealed by changes in trypsinization pattern.

Repetitive homogenization of skeletal muscle sarcoplasmic reticulum (SR) membranes in the presence of chelating agents at low ionic strength leads to the loss of the Ca-ATPase activity. This inactive state of the enzyme is coupled to an extensive rearrangement of the cytosolic domains as visualized by a completely different trypsinization pattern of the enzyme. In addition to the primary cleavage site (Arg 505), a novel trypsinization site (Arg 334), just N-terminal of the phosphorylation domain and localized on the primary tryptic fragment A, becomes exposed. Cleavage at the latter site yields a soluble fragment of M(r) 20,117 and the membrane-bound N-terminal one-third of the ATPase of M(r) 35,279. Two additional trypsinization sites C-terminal of the nucleotide binding domain become exposed in the inactive Ca(2+)-ATPase conformation. Rapid cleavage at these sites yields two soluble fragments of about 15 and 10 kDa. All together, the three soluble fragments comprise most of the large cytosolic loop of the Ca(2+)-ATPase. The inactivation and the change in trypsinization pattern can be reversed by rehomogenization of the extracted membranes in the presence of divalent cations. The results suggest the presence of an occluded site for divalent cations which can be depleted or refilled during application of sheer forces. Occupation of this site is essential to confer to the enzyme an active conformation.

Characterization of the potassium channel from frog skeletal muscle sarcoplasmic reticulum membrane.

1. The sarcoplasmic reticulum (SR) membrane of skeletal muscle contains potassium channels which are thought to support charge neutralization during calcium release by providing a permeability pathway for counter-ion movement. To describe the behaviour of the SR K+ channel under physiological conditions, single channel activity was recorded from excised patches of SR membrane. Patches were made from membrane blebs extruded from contracted muscle fibres whose surface membranes had been removed previously by mechanical dissection. 2. The channel was active over a large voltage range from -80 to +100 mV. The current-voltage relationship of the channel was linear over most of this voltage range (slope conductance equal to 60 pS in 130 mM potassium), but showed rectification at voltages below -50 mV. 3. The activity of the channel (number of state transitions per unit time) was greater at positive voltages than at negative voltages. Analysis of dwell-time distributions showed that the time spent in the open state is best fitted by a double Gaussian, suggesting that the channel possesses both a long (l)- and a short (s)-lived open state with identical conductances. The dwell times for the two states were Ts = 0.3 ms and Tl = 2.6 ms at +90 mV and Ts = 0.1 ms and Tl = 15.1 ms at -40 mV. Thus, positive voltage decreased the long open time significantly which was consistent with the observed increase in channel activity at positive potentials. 4. The permeability sequence of the channel to various monovalent cations was deduced from the channel reversal potential under bi-ionic conditions and was found to be: K+ > Rb+ > Na+ > Cs+ > Li+. 5. Channel activity was reduced when the patch was perfused with 1,10-bis-guanidino-n-decane (BisG10), a drug reported to block the SR K+ channel with high affinity. The drug concentration necessary to reduce the open probability (P(o)) by 50% was 19.8 microM at -40 mV and 338.2 microM at +50 mV. The zero voltage dissociation constant (Kd) was calculated to be 48 microM. 6. Pharmacological agents known to affect surface membrane K+ channels, such as 0.5 mM Ba2+ or 3.0 mM 4-aminopyridine, were much less effective in blocking the channel than BisG10. Physiological calcium concentrations (pCa = 8.0 and 3.0) did not affect channel behaviour.4

Modulation of Cardiac Sarcoplasmic Reticulum Ryanodine Receptor by Sphingosine

Excitation contraction (EC) coupling in muscle cells involves the movement of calcium through the calcium release channel of the sarcoplasmic reticulum (SR) membrane known as the ryanodine receptor. We have recently shown that the novel second messenger, sphingosine, can block calcium release from skinned skeletal muscle fibers and from isolated skeletal muscle SR membranes (Sabbadini et al., J Biol Chem 267; 15475-15484, 1992). In this report, we demonstrate that sphingosine also inhibits calcium release from isolated canine cardiac SR membranes containing the ryanodine receptor when release is induced by caffeine, doxorubicin or by calcium. Sphingosine also prevents the augmentation of [3H]-ryanodine binding normally produced by caffeine and doxorubicin and exerts noncompetitive inhibition with regard to both releasing agents. Sphingosine significantly reduces in a dose-dependent manner [3H]-ryanodine binding to the high affinity site of the receptor and increases by several-fold the Kd for binding, which is consistent with a blocking action of sphingosine on the ryanodine receptor calcium channel. Sphingosine inhibits the extent of calcium-induced calcium release (CICR) and significantly shifts the threshold for CICR so that a higher level of trigger calcium is required to initiate CICR. The sphingosine inhibition of CICR is consistent with the near abolition of calcium dependent [3H]-ryanodine binding. HPLC analysis of cardiac sphingosine content indicates that sphingosine is present in the cardiac cell at moderately high levels (29.4 nmol/g wet wt for the entire cell and ∼0.4 μM for the cytosol) which are sufficient to produce significant inhibition by sphingosine on calcium release and ryanodine binding. The data suggest that sphingosine acts on the cardiac ryanodine receptor by opposing the physiological stimulus (e.g. trigger calcium entering via the dihydropyridine receptor). We propose that sphingosine is produced by the T-tubule membranes and that sphingosine is released into the protected intracellular environment of the T-tubule/SR junction to negatively modulate calcium release. Consequently, it is possible that sphingosine is a physiologically relevant regulator of calcium levels in the heart.

Functional coupling between sarcoplasmic-reticulum-bound creatine kinase and Ca(2+)-ATPase.

We investigated the role of creatine kinase bound to sarcoplasmic reticulum membranes of fast skeletal muscle in the local regeneration of ATP and the possible physiological significance of this regeneration for calcium pump function. Our results indicate that ADP produced by sarcoplasmic reticulum Ca(2+)-ATPase is effectively phosphorylated by creatine kinase in the presence of creatine phosphate. This phosphorylation is an important function of the membrane-bound creatine kinase because accumulation of ADP has a depressive effect on Ca(2+)-uptake by sarcoplasmic reticulum vesicles. The concentration-dependent depression of Ca(2+)-uptake by ADP was especially pronounced when there was strong back inhibition by high intravesicular [Ca2+]. ATP regenerated by endogenous creatine kinase was not in free equilibrium with the ATP in the surrounding medium, but was used preferentially by Ca(2+)-ATPase for Ca(2+)-uptake. Efficient translocation of ATP from creatine kinase to Ca(2+)-ATPase, despite the presence of an ATP trap in the surrounding medium, can be explained by close localization of creatine kinase and Ca(2+)-ATPase on the sarcoplasmic reticulum membranes. These results suggest the existence of functional coupling between creatine kinase and Ca(2+)-ATPase on skeletal muscle sarcoplasmic reticulum membranes. Several factors (amount of membrane-bound creatine kinase, oxidation of SH groups of creatine kinase, decrease in [phosphocreatine]) can influence the ability of creatine kinase/phosphocreatine system to support a low ADP/ATP ratio and fuel the Ca(2+)-pump with ATP. These factors may become operative in the living cells, influencing functional coupling between creatine kinase and Ca(2+)-ATPase and may have an indirect effect on Ca(2+)-pump function before Ca(2+)-ATPase itself is affected.

Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase.

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.

The influence of Mg 2+ on anion binding to sarcoplasmic reticulum membranes as detected by 35Cl-NMR

35Cl-NMR spectroscopy has been used to study the competition between anions, including nucleotides, on skeletal muscle sarcoplasmic reticulum membranes. Different chloride binding sites can be distinguished according to their Mg 2+ sensitivity. Phosphate binding is enhanced by Mg 2+ whereas the anion transport inhibitor pyridoxalphosphate-6-azophenyl-2′-sulfonic acid (PPAPS) binding is not. The affinity of the enzyme for the Mg-adenylyl imidodiphosphate (MgAMP-PNP) complex is decreased whereas that for MgATP is increased. Three sets of binding sites can be discriminated from which chloride is displaced by different anions with varying efficiency. High affinity binding of AMP-PNP and PPAPS occurs at the same site, that can also be occupied by phosphate. Low-affinity binding of PPAPS and AMP-PNP also coincides, but in a site where phosphate binding is negligible. ATP and ADP bind to both sites. In the presence of Mg 2+ a third anion binding site can be occupied by phosphate but neither by AMP-PNP nor PPAPS.

Inhibition of dicarboxylic anion transport by fluorescein isothocyanate in skeletal sarcoplasmic reticulum

It has been demonstrated previously that dicarboxylic anions are cotransported during ATP-dependent Ca 2+ transport by skeletal muscle sarcoplasmic reticulum (SR) membranes, and that anion cotransport stimulates Ca 2+ transport. In the current study, we present evidence that dicarboxylic anion cotransport and Ca 2+ transport are kinetically distinct in SR, but both functions are mediated by the CaATPase protein. Preincubation of SR with 40 μ m fluorescein isothiocyanate (FITC) (pH 7.0) inhibited essentially all of the Ca 2+ ATPase activity, as well as active oxalate-supported and oxalate-independent 45Ca 2+ accumulation. The addition of 1 m m β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) to the preincubation media fully protected the dicarboxylic anion-independent Ca 2+ ATPase activity and the oxalate-independent active 45Ca 2+ accumulation from the inhibitory effects of FITC; however, the ATP-associated [ 14C]oxalate accumulation, the oxalate-dependent 45Ca 2+ accumulation, and the oxalate- and maleate-dependent stimulation of Ca 2+ ATPase activity were not protected by AMP-PCP. Thus, the dicarboxylic anion accumulation and the stimulation of Ca 2+ uptake by dicarboxylic anions could be functionally separated from the ATP-dependent, anion-independent Ca 2+ translocation. FITC bound exclusively to the 100-kDa (CaATPase) and 92-kDa (phosphorylase) proteins in the SR membranes and to purified CaATPase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 1 m m AMP-PCP inhibited 50–55% of the FITC fluorescence on the 100-kDa protein, but did not significantly alter fluorescence on the 92-kDa protein. Two-dimensional gel analysis demonstrated a single 100-kDa protein in longitudinal SR membranes. FITC appears to inhibit ATP-dependent Ca 2+ transport, and dicarboxylic anion translocation through interaction at separate domains of the CaATPase protein.

Influence of the 53 kDa glycoprotein on the cooperativity of the Ca 2+-ATPase of the sarcoplasmic reticulum

Previous results from this laboratory suggest that the 53 kDa glycoprotein (GP-53) of rabbit skeletal muscle sarcoplasmic reticulum membrane (SR) may influence coupling between Ca 2+ transport and ATP hydrolysis by the Ca 2+-ATPase. Here we report evidence that GP-53 may influence the cooperative behavior of the Ca 2+-ATPase. The ATPase activity of the Ca 2+-ATPase displays negative cooperative dependence (Hill coefficient n<1) on [MgATP] and has positive cooperative dependence ( n>1) on [Ca 2+] free. We have determined the degree of cooperativity for native SR vesicles, SR preincubated with antiserum against GP-53 or preimmume serum, and SR partially extracted with KCl-cholate. Our results show that SR preincubated with preimmune serum or SR treated with cholate in 50 mM KCl (yielding membranes rich in GP-53) demonstrate a coperative dependence of Ca 2+-ATPase activity on both [ATP] and [Ca 2+] similar to that of untreated SR. SR preincubated with anti-GP-53 antiserum (which causes an uncoupling of Ca 2+ transport from ATP hydrolysis) or SR extracted with cholate in 1 M KCl (yielding membranes depleted of GP-53) displays decreased positive cooperative dependence on [Ca 2+] and decreased negative cooperative dependence on [ATP]. The results are consistent with the interpretation that GP-53 may influence the cooperative behavior of the Ca 2+-ATPase.

Effect of halothane on human skeletal muscle sarcoplasmic reticulum calcium-release channel.

In order to gain an understanding of how anesthetics alter muscle function, we have utilized the planar lipid bilayer technique for recording the conductance and gating properties of a calcium-release channel molecule derived from native skeletal muscle sarcoplasmic reticulum membranes. The incorporation of this protein molecule into an artificial membrane simplifies investigation, limiting anesthetic action to the bilayer itself and/or the single protein molecule. Thus, any effect the anesthetic has on the protein’s conductance and gating functions will be a consequence of the anesthetic’s action directly on the protein and/or on the lipid bilayer in such a way as to alter the protein’s function. Our attempts to understand a piece of the puzzle of anesthetic action on skeletal muscle do, in fact, begin at the molecular, single protein level of attack and even so, this experimental model is far from simple, and the possible ways by which volatile anesthetics could alter its function represent a challenge. Our piece of the puzzle involves a large protein molecule located in the structure bridging the transverse tubule and the terminal cisternae of the sarcoplasmic reticulum (see Chapter 1, figure 1). This protein, sometimes referred to as the ryanodine receptor protein, is thought to play a significant role in excitation-contraction coupling of skeletal muscle. In our present studies, we have incorporated this protein into a planar lipid bilayer and have recorded and measured its properties as a cation-conducting channel. These studies parallel those on the pharmacogenetic disease malignant hyperthermia (MH) and illustrate how such a disease may lead to a better understanding of normal muscle response to volatile anesthetics.