Effect of lactate on depolarization-induced Ca(2+) release in mechanically skinned skeletal muscle fibers

Itis unclear whether accumulation of lactate in skeletal muscle fibersduring intense activity contributes to muscle fatigue. Usingmechanically skinned fibers from rat and toad muscle, we were able toexamine the effect of L(+)-lactate onexcitation-contraction coupling independently of other metabolicchanges. We investigated the effects of lactate on the contractileapparatus, caffeine-induced Ca²⁺ release from thesarcoplasmic reticulum, and depolarization-induced Ca²⁺release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable(20-35%) inhibition of caffeine-induced Ca²⁺ release,seemingly by a direct effect on the Ca²⁺ release channels.However, 15 mM lactate had no detectable effect on Ca²⁺release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect onnormal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.

     

Two domains in dihydropyridine receptor activate the skeletal muscle Ca(2+) release channel

The II-III cytoplasmic loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1)-subunit is essential for skeletal-type excitation-contraction coupling. Single channel and [(3)H]ryanodine binding studies with a full-length recombinant peptide (p(666-791)) confirmed that this region specifically activates skeletal muscle Ca2+ release channels (CRCs). However, attempts to identify shorter domains of the II-III loop specific for skeletal CRC activation have yielded contradictory results. We assessed the specificity of the interaction of five truncated II-III loop peptides by comparing their effects on skeletal and cardiac CRCs in lipid bilayer experiments; p(671-680) and p(720-765) specifically activated the submaximally Ca2+-activated skeletal CRC in experiments using both mono and divalent ions as current carriers. A third peptide, p(671-690), showed a bimodal activation/inactivation behavior indicating a high-affinity activating and low-affinity inactivating binding site. Two other peptides (p(681-690) and p(681-685)) that contained an RKRRK-motif and have previously been suggested in in vitro studies to be important for skeletal-type E-C coupling, failed to specifically stimulate skeletal CRCs. Noteworthy, p(671-690), p(681-690), and p(681-685) induced similar subconductances and long-lasting channel closings in skeletal and cardiac CRCs, indicating that these peptides interact in an isoform-independent manner with the CRCs.     

Effects of cytoplasmic and luminal pH on Ca(2+) release channels from rabbit skeletal muscle

Ryanodine receptor (RyR)-Ca(2+) release channels from rabbit skeletal muscle were incorporated into lipid bilayers. The effects of cytoplasmic and luminal pH were studied separately over the pH range 5-8, using half-unit intervals. RyR activity (at constant luminal pH of 7.5) was inhibited at acidic cytoplasmic pH, with a half-inhibitory pH (pH(I)) approximately 6.5, irrespective of bilayer potential and of whether the RyRs were activated by cytoplasmic Ca(2+) (50 microM), ATP (2 or 5 mM), or both. Inhibition occurred within approximately 1 s and could be fully reversed within approximately 1 s after brief inhibition or within approximately 30-60 s after longer exposure to acidic cytosolic pH. There was no evidence of any hysteresis in the cytoplasmic pH effect. Ryanodine-modified channels were less sensitive to pH inhibition, with pH(I) at approximately 5.5, but the inhibition was similarly reversible. Steady-state open and closed dwell times of RyRs during cytoplasmic pH inhibition suggest a mechanism where the binding of one proton inhibits the channel and the binding of two to three additional protons promotes further inhibited states. RyR activity was unaffected by luminal pH in the pH range 7.5 to 6.0. At lower luminal pH (5-5.5) most RyRs were completely inhibited, and raising the pH again produced partial to full recovery in only approximately 50% of cases, with the extent of recovery not detectably different between pH 7.5 and pH 9. The results indicate that isolated skeletal muscle RyRs are not inhibited as strongly by low cytoplasmic and luminal pH, as suggested by previous single-channel studies.     

Ca(2+)-dependent interaction between FKBP12 and calcineurin regulates activity of the Ca(2+) release channel in skeletal muscle

Calcineurin is a Ca(2+) and calmodulin-dependent protein phosphatase with diverse cellular functions. Here we examined the physical and functional interactions between calcineurin and ryanodine receptor (RyR) in a C2C12 cell line derived from mouse skeletal muscle. Coimmunoprecipitation experiments revealed that the association between RyR and calcineurin exhibits a strong Ca(2+) dependence. This association involves a Ca(2+) dependent interaction between calcineurin and FK506-binding protein (FKBP12), an accessory subunit of RyR. Pretreatment with cyclosporin A, an inhibitor of calcineurin, enhanced the caffeine-induced Ca(2+) release (CICR) in C2C12 cells. This effect was similar to those of FK506 and rapamycin, two drugs known to cause dissociation of FKBP12 from RyR. Overexpression of a constitutively active form of calcineurin in C2C12 cells, DeltaCnA(391-521) (deletion of the last 131 amino acids from calcineurin), resulted in a decrease in CICR. This decrease in CICR activity was partially recovered by pretreatment with cyclosporin A. Furthermore, overexpression of an endogenous calcineurin inhibitor (cain) or an inactive form of calcineurin (DeltaCnA(H101Q)) in C2C12 cells resulted in up-regulation of CICR. Taken together, our data suggest that a trimeric-interaction among calcineurin, FKBP12, and RyR is important for the regulation of the RyR channel activity and may play an important role in the Ca(2+) signaling of muscle contraction and relaxation.     

Effects of dihydropyridine receptor II-III loop peptides on Ca(2+) release in skinned skeletal muscle fibers

Inskeletal muscle fibers, the intracellular loop between domains II andIII of the ₁-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca²⁺ releasechannel in the sarcoplasmic reticulum. We examined the effects ofsynthetic peptide segments of this loop on Ca²⁺ release inmechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg²⁺ concentration ([Mg²⁺]; 1 mM), a20-residue skeletal muscle DHPR peptide[A_S(20);Thr⁶⁷¹-Leu⁶⁹⁰; 30 µM], shown previously toinduce Ca²⁺ release in a triad preparation, caused onlysmall spontaneous force responses in ~40% of fibers, although itpotentiated responses to depolarization and caffeine in all fibers. TheCOOH-terminal half of A_S(20)[A_S(10)] induced much larger spontaneousresponses but also caused substantial inhibition of Ca²⁺release to both depolarization and caffeine. Both peptides induced orpotentiated Ca²⁺ release even when the voltage sensors wereinactivated, indicating direct action on the Ca²⁺ releasechannels. The corresponding 20-residue cardiac DHPR peptide [A_C(20);Thr⁷⁹³-Ala⁸¹²] was ineffective, but itsCOOH-terminal half [A_C(10)] had effects similar to A_S(20). In the presence of lower[Mg²⁺] (0.2 mM), exposure to eitherA_S(20) or A_C(10) (30 µM) induced substantial Ca²⁺ release. PeptideC_S (100 µM), a loop segment reported to inhibit Ca²⁺ release in triads, caused partial inhibition ofdepolarization-induced Ca²⁺ release. In toad fibers, eachof the A peptides had effects similar to or greater than those in ratfibers. These findings suggest that the A and C regions of the skeletalDHPR II-III loop may have important roles in vivo.

     

A negatively charged region of the skeletal muscle ryanodine receptor is involved in Ca(2+)-dependent regulation of the Ca(2+) release channel

The ryanodine receptor/Ca(2+) release channels from skeletal (RyR1) and cardiac (RyR2) muscle cells exhibit different inactivation profiles by cytosolic Ca(2+). D3 is one of the divergent regions between RyR1 (amino acids (aa) 1872-1923) and RyR2 (aa 1852-1890) and may contain putative binding site(s) for Ca(2+)-dependent inactivation of RyR. To test this possibility, we have deleted the D3 region from RyR1 (DeltaD3-RyR1), residues 1038-3355 from RyR2 (Delta(1038-3355)-RyR2) and inserted the skeletal D3 into Delta(1038-3355)-RyR2 to generate sD3-RyR2. The channels formed by DeltaD3-RyR1 and Delta(1038-3355)-RyR2 are resistant to inactivation by mM [Ca(2+)], whereas the chimeric sD3-RyR2 channel exhibits significant inactivation at mM [Ca(2+)]. The DeltaD3-RyR1 channel retains its sensitivity to activation by caffeine, but is resistant to inactivation by Mg(2+). The data suggest that the skeletal D3 region is involved in the Ca(2+)-dependent regulation of the RyR1 channel.     

Reduced Ca(2+)-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum at low pH.

The purpose of this investigation was to determine the effects of reduced pH on Ca(2+)-induced Ca2+ release (CICR) from skeletal muscle sarcoplasmic reticulum (SR). Frog semitendinosus fiber bundles (1-3/bundle) were chemically skinned via saponin treatment (50 micrograms/mL, 20 min), which removes the sarcolemma and leaves the SR functional. The SR was first depleted of Ca2+ then loaded for 2 min at pCa (log free Ca2+ concentration) 6.6. CICR was then evoked by exposing the fibers to pCa 5-7 for 5-60 s. CICR was evoked both in the absence of ATP and Mg2+ and in the presence of beta, gamma-methyleneadenosine-5'-triphosphate (AMPPCP, a nonhydrolyzable form of ATP) and Mg2+. Ca2+ remaining in the SR was then assayed via caffeine (25 mM) contracture. In all cases, CICR evoked at pH 6.5 resulted in larger caffeine contractures than that evoked at 7.0, suggesting that more Ca2+ was released during CICR at the higher pH. Accordingly, rate constants for CICR were significantly greater at pH 7.0 than at pH 6.5. These results indicate that reduced pH depresses CICR from skeletal muscle SR.     

Mechanisms of P(i) regulation of the skeletal muscle SR Ca(2+) release channel

Inorganic phosphate(P_i) accumulates in the fibers of actively working musclewhere it acts at various sites to modulate contraction. To characterizethe role of P_i as a regulator of the sarcoplasmic reticulum(SR) calcium (Ca²⁺) release channel, we examined the actionof P_i on purified SR Ca²⁺ release channels,isolated SR vesicles, and skinned skeletal muscle fibers. In singlechannel studies, addition of P_i to the cis chamberincreased single channel open probability (P_o;0.079 ± 0.020 in 0 P_i, 0.157 ± 0.034 in 20 mMP_i) by decreasing mean channel closed time; mean channelopen times were unaffected. In contrast, the ATP analog,,-methyleneadenosine 5'-triphosphate (AMP-PCP), enhancedP_o by increasing single channel open time anddecreasing channel closed time. P_i stimulation of[³H]ryanodine binding by SR vesicles wassimilar at all concentrations of AMP-PCP, suggesting P_i andadenine nucleotides act via independent sites. In skinned musclefibers, 40 mM P_i enhanced Ca²⁺-inducedCa²⁺ release, suggesting an in situ stimulation ofthe release channel by high concentrations of P_i. Ourresults support the hypothesis that P_i may be an importantendogenous modulator of the skeletal muscle SR Ca²⁺ releasechannel under fatiguing conditions in vivo, acting via a mechanismdistinct from adenine nucleotides.

     

Ca2+ release by inositol-trisphosphorothioate in isolated triads of rabbit skeletal muscle.

The effectiveness of the nonmetabolizable second messenger analogue DL-myo-inositol 1,4,5-trisphosphorothioate (IPS3) described by Cooke, A. M., R. Gigg, and B. V. L. Potter, (1987b. Jour. Chem. Soc. Chem. Commun. 1525-1526.) was examined in triads purified from rabbit skeletal muscle. A Ca2+ electrode uptake-release assay was used to determine the size and sensitivity of the IPS3-releasable pool of Ca2+ in isolated triads. Uptake was initiated by 1 mM MgATP, pCa 5.8, pH 7.5 Release was initiated when the free Ca2+ had lowered to pCa approximately 7. We found that 5-25 microM myo-inositol 1,4,5-trisphosphate (IP3), and separately IPS3, consistently released 5-20% of the Ca2+ pool actively loaded into triads. Single channel recording was used to determine if ryanodine receptor Ca2+ release channels were affected by IPS3 at the same myoplasmic Ca2+ and IPS3 concentrations. Open probability of ryanodine receptor Ca2+ release channels was monitored in triads fused to bilayers over long periods (200 s) in the absence and following addition of 30 microM IPS3 to the same channel. At myoplasmic pCa approximately 7, IPS3 had no effect in the absence of MgATP (Po = 0.0094 +/- 0.001 in control and Po = 0.01 +/- 0.006 after IPS3) and slightly increased activity in the presence of 1 mM MgATP (Po = 0.024 +/- 0.03 in control and Po = 0.05 +/- 0.03 after IPS3). Equally small effects were observed at higher myoplasmic Ca2+. The onset of channel activation by IPS3 or IP3 was slow, on the time scale 20-60 s. We suggest that in isolated triads of rabbit skeletal muscle, IP3-induced release of stored Ca2+ is probably not mediated by the opening of Ca2+ release channels.     

(Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles

We have investigated some characteristics of the sarcoplasmic reticulum (Ca2+ + Mg2+)-dependent ATPase (Ca2+-ATPase) mRNA from smooth muscle using specific cDNA probes isolated from a rat heart cDNA library. RNA blot analysis has shown that the Ca2+-ATPase mRNA expressed in smooth muscle is identical in size to the cardiac mRNA but differs from that of fast skeletal muscle. S1 nuclease mapping has moreover shown that the cardiac and smooth muscle isoforms possess different 3'-end sequences. These results indicate that a distinct sarcoplasmic reticulum Ca2+-ATPase mRNA is present in smooth muscle.     

Voltage-independent changes in L-type Ca(2+) current uncoupled from SR Ca(2+) release in cardiac myocytes

To determine the effect of voltage-independent alterations of L-type Ca(2+) current (I(Ca)) on the sarcoplasmic reticular (SR) Ca(2+) release in cardiac myocytes, we measured I(Ca) and cytosolic Ca(2+) transients (Ca(i)(2+); intracellular Ca(2+) concentration) in voltage-clamped rat ventricular myocytes during 1) an abrupt increase of extracellular [Ca(2+)] (Ca(o)(2+)) or 2) application of 1 microM FPL-64176, a Ca(2+) channel agonist, to selectively alter I(Ca) in the absence of changes in SR Ca(2+) loading. On the first depolarization in higher Ca(o)(2+), peak I(Ca) was increased by 46 +/- 6% (P < 0.001), but the increases in the maximal rate of rise of Ca(i)(2+) (dCa(i)(2+)/dt(max), where t is time; an index of SR Ca(2+) release flux) and the Ca(i)(2+) transient amplitude were not significant. Rapid exposure to FPL-64176 greatly slowed inactivation of I(Ca), increasing its time integral by 117 +/- 8% (P < 0.001) without significantly increasing peak I(Ca), dCa(i)(2+)/dt(max), or amplitude of the corresponding Ca(i)(2+) transient. Prolongation of exposure to higher Ca(o)(2+) or FPL-64176 did not further increase peak I(Ca) but greatly increased dCa(i)(2+)/dt(max), Ca(i)(2+) transient amplitude, and the gain of Ca(2+) release (dCa(i)(2+)/dt(max)/I(Ca)), evidently due to augmentation of the SR Ca(2+) loading. Also, the time to peak dCa(i)(2+)/dt(max) was significantly increased in the continuous presence of higher Ca(o)(2+) (by 37 +/- 5%, P < 0.001) or FPL-64176 (by 63 +/- 5%, P < 0.002). Our experiments provide the first evidence of a marked disparity between an increased peak I(Ca) and the corresponding SR Ca(2+) release. We attribute this to saturation of the SR Ca(2+) release flux as predicted by local control theory. Prolongation of the SR Ca(2+) release flux, caused by combined actions of a larger I(Ca) and maximally augmented SR Ca(2+) loading, might reflect additional Ca(2+) release from corbular SR.     

H-NMR study of rabbit skeletal muscle troponin C: Ca(2+)-dependent interaction with mastoparan.

1H-NMR spectroscopy is employed to study the interaction between rabbit skeletal muscle troponin (C (TnC) and wasp venom tetradecapeptide mastoparan. We monitored the spectral change of the following species of TnC as a function of mastoparan concentration: apoTnC, Ca(2+)-saturated TnC (Ca4TnC) and Ca(2+)-half loaded TnC (Ca2TnC). When apo-TnC is titrated with mastoparan, line-broadening is observed for the ring-current shifted resonance of Phe-23, Ile-34, Val-62 and Phe-72 and the downfield-shifted CH alpha-resonances of Asp-33, Thr-69 and Asp-71; these residues are located in the N-domain. When Ca4TnC is titrated with mastoparan, chemical shift change is observed for the ring-current shifted resonances of Phe-99, Ile-110 and Phe-148 and the downfield-shifted CH alpha-resonances of Asn-105, Ala-106, Ile-110 and Ile-146 and aromatic resonance of Tyr-109 and His-125; these residues are located in the C-domain. The resonance of Phe-23, Asp-33, Asp-71, Phe-72, Phe-99, Tyr-109, Ile-146, His-125 and Phe-148 in both N- and C-domains changes when Ca2TnC is titrated with mastoparan. These results suggest that mastoparan binds to the N-domain of apo-TnC, the C-domain of Ca4TnC and the N- and C-domains of Ca2TnC; the hydrophobic cluster in each domain is involved in binding. As mastoparan binds to TnC, the above resonances shift to their normal chemical shift positions. The stability of the cluster and the beta-sheet is reduced by mastoparan-binding. These results suggest that the conformation of the hydrophobic cluster and the neighboring beta-sheet change to a loose form. The stability of the N-domain of Ca2TnC and Ca4TnC increases when these species bind 1 mol of mastoparan at the C-domain. These results suggest a mastoparan-induced interaction between the N- and C-domains of TnC.     

Voltage clamp methods for the study of membrane currents and SR Ca(2+) release in adult skeletal muscle fibres

Skeletal muscle excitation-contraction (E-C)(1) coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)(2) Ca(2+) release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca(2+), due to depolarization-initiated SR Ca(2+) release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or 'high resistance gap' techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca(2+) signalling properties of mouse skeletal muscle membranes are being investigated.     

Ca(2+)-dependent proteolysis in muscle wasting

Skeletal muscle wasting is a prominent feature of cachexia, a complex systemic syndrome that frequently complicates chronic diseases such as inflammatory and autoimmune disorders, cancer and AIDS. Muscle wasting may also develop as a manifestation of primary or neurogenic muscular disorders. It is now generally accepted that muscle depletion mainly arises from increased protein catabolism. The ubiquitin-proteasome system is believed to be the major proteolytic machinery in charge of such protein breakdown, yet there is evidence suggesting that Ca(2+)-dependent system, lysosomes and, in some conditions at least, even caspases are involved as well. The role of Ca(2+)-dependent proteolysis in skeletal muscle wasting is reviewed in the present paper. This system relies on the activity of calpains, a family of Ca(2+)-dependent cysteine proteases, whose regulation is complex and not completely elucidated. Modulations of Ca(2+)-dependent proteolysis have been associated with muscle protein depletion in various pathological contexts and particularly with muscle dystrophies. Calpains can only perform a limited proteolysis of their substrates, however they may play a critical role in initiating the breakdown of myofibrillar protein, by releasing molecules that become suitable for further degradation by proteasomes. Some evidence would also support a role for lysosomes and caspases in muscle wasting. Thus it cannot be excluded that different intracellular proteolytic systems may coordinately concur in shifting muscle protein turnover towards excess catabolism. Many different signals have been proposed as potentially involved in triggering the enhanced protein breakdown that underlies muscle wasting. How they are transduced to initiate the hypercatabolic response and to activate the proteolytic pathways remains largely unknown, however.     

DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers

Changes in skeletal muscle volume induce localized sarcoplasmic reticulum (SR) Ca²⁺ release (LCR) events, which are sustained for many minutes, suggesting a possible signaling role in plasticity or pathology. However, the mechanism by which cell volume influences SR Ca²⁺ release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrode''s solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (E_m), and intracellular Ca²⁺ ([Ca²⁺]_i) were determined. To allow comparison with previous studies, solutions were made hyperosmotic by the addition of sugars or divalent cations, or they were made hypoosmotic by reducing [NaCl]_o. All hyperosmotic solutions induced a sustained decrease in cell volume, which was accompanied by membrane depolarization (by 14–18 mV; n = 40) and SR Ca²⁺ release. However, sugar solutions caused a global increase in [Ca²⁺]_i, whereas solutions made hyperosmotic by the addition of divalent cations only induced LCR. Decreasing osmolarity induced an increase in cell volume and a negative shift in E_m (by 15.04 ± 1.85 mV; n = 8), whereas [Ca²⁺]_i was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and E_m was associated with LCR. Both global and localized SR Ca²⁺ release were abolished by the dihydropyridine receptor inhibitor nifedipine by sustained depolarization of the sarcolemmal or by the addition of the ryanodine receptor 1 inhibitor tetracaine. Inhibitors of the Na-K-2Cl (NKCC) cotransporter markedly inhibited the depolarization associated with hyperosmotic shrinkage and the associated SR Ca²⁺ release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca²⁺ release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in E_m. The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions.     

Characteristics of phosphate-induced Ca(2+) efflux from the SR in mechanically skinned rat skeletal muscle fibers

ATP is a candidate enteric inhibitory neurotransmitterin visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca²⁺-activatedK⁺ (SK) channels. Coupling between ATP stimulation and SKchannels may be mediated by localized Ca²⁺ release.Isolated myocytes of the murine colon produced spontaneous, localizedCa²⁺ release events. These events corresponded tospontaneous transient outward currents (STOCs) consisting ofcharybdotoxin (ChTX)-sensitive and -insensitive events.ChTX-insensitive STOCs were inhibited by apamin. LocalizedCa²⁺ transients were not blocked by ryanodine, but theseevents were reduced in magnitude and frequency by xestospongin C(Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus wehave termed the localized Ca²⁺ events in colonic myocytes"Ca²⁺ puffs." The P_2Y receptor agonist2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency ofCa²⁺ puffs. 2-MeS-ATP also increased STOCs in associationwith the increase in Ca²⁺ puffs.Pyridoxal-phospate-6-azophenyl-2',4'-disculfonic acid tetrasodium, aP₂ receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca²⁺ transients and the effects of 2-MeS-ATP onCa²⁺ puffs and STOCs were blocked by U-73122, an inhibitorof phospholipase C. Xe-C and ryanodine also blocked responses to2-MeS-ATP, suggesting that, in addition to release from IP₃receptor-operated stores, ryanodine receptors may be recruited duringagonist stimulation to amplify release of Ca²⁺. These datasuggest that localized Ca²⁺ release modulatesCa²⁺-dependent ionic conductances in the plasma membrane.Localized Ca²⁺ release may contribute to the electricalresponses resulting from purinergic stimulation.

     

RyR3 amplifies RyR1-mediated Ca(2+)-induced Ca(2+) release in neonatal mammalian skeletal muscle

The neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane. An allosteric interaction between RyR1 and dihydropyridine receptors located in the plasma membrane mediates voltage-induced Ca(2+) release (VICR) from the sarcoplasmic reticulum. RyR3, which disappears in adult muscle, is not involved in VICR, and the role of the transiently expressed RyR3 remains elusive. Here we demonstrate that RyR1 participates in both VICR and Ca(2+)-induced Ca(2+) release (CICR) and that RyR3 amplifies RyR1-mediated CICR in neonatal skeletal muscle. Confocal measurements of intracellular Ca(2+) in primary cultured mouse skeletal myotubes reveal active sites of Ca(2+) release caused by peripheral coupling between dihydropyridine receptors and RyR1. In myotubes lacking RyR3, the peripheral VICR component is unaffected, and RyR1s alone are able to support inward CICR propagation in most cells at an average speed of approximately 190 microm/s. With the co-presence of RyR1 and RyR3 in wild-type cells, unmitigated radial CICR propagates at 2,440 microm/s. Because neonatal skeletal muscle lacks a well developed transverse tubule system, the RyR3 reinforcement of CICR seems to ensure a robust, uniform, and synchronous activation of Ca(2+) release throughout the cell body. Such functional interplay between RyR1 and RyR3 can serve important roles in Ca(2+) signaling of cell differentiation and muscle contraction.     

o-Phthalaldehyde activates the Ca(2+) release mechanism from skeletal muscle sarcoplasmic reticulum.

o-Phthalaldehyde (OPA) is a bifunctional reagent that forms an isoindole derivative by reacting with cysteine and lysine residues separated by approximately 0.3 nm. OPA inhibits sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity at low micromolar concentrations and induces Ca(2+) release from actively loaded SR vesicles by activating the ryanodine receptor from fast twitch skeletal muscle. Both ryanodine binding and single-channel activity show a biphasic concentration dependence. At low OPA concentrations (<100 microM), ryanodine binding and single channel activity are stimulated, while at higher concentrations, a time-dependent sequential activation and inhibition of receptor binding is observed. Activation is characterized by a Ca(2+)-independent increase in maximal receptor occupancy. Data are presented to support a model in which Ca(2+) channel and ryanodine binding activity are enhanced due to an intramolecular cross-linking of nearby lysine and nonhyperreactive cysteine residues. OPA complexation with endogenous lysine residue(s) is critical for receptor activation.     

Measurement of RyR permeability reveals a role of calsequestrin in termination of SR Ca(2+) release in skeletal muscle

The mechanisms that terminate Ca(2+) release from the sarcoplasmic reticulum are not fully understood. D4cpv-Casq1 (Sztretye et al. 2011. J. Gen. Physiol. doi:10.1085/jgp.201010591) was used in mouse skeletal muscle cells under voltage clamp to measure free Ca(2+) concentration inside the sarcoplasmic reticulum (SR), [Ca(2+)](SR), simultaneously with that in the cytosol, [Ca(2+)](c), during the response to long-lasting depolarization of the plasma membrane. The ratio of Ca(2+) release flux (derived from [Ca(2+)](c)(t)) over the gradient that drives it (essentially equal to [Ca(2+)](SR)) provided directly, for the first time, a dynamic measure of the permeability to Ca(2+) of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, [Ca(2+)](SR) stabilized at ～35% of its resting level; depletion was therefore incomplete. By 0.4 s of depolarization, the measured permeability decayed to ～10% of maximum, indicating ryanodine receptor channel closure. Inactivation of the t tubule voltage sensor was immeasurably small by this time and thus not a significant factor in channel closure. In cells of mice null for Casq1, permeability did not decrease in the same way, indicating that calsequestrin (Casq) is essential in the mechanism of channel closure and termination of Ca(2+) release. The absence of this mechanism explains why the total amount of calcium releasable by depolarization is not greatly reduced in Casq-null muscle (Royer et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.201010454). When the fast buffer BAPTA was introduced in the cytosol, release flux became more intense, and the SR emptied earlier. The consequent reduction in permeability accelerated as well, reaching comparable decay at earlier times but comparable levels of depletion. This observation indicates that [Ca(2+)](SR), sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates Ca(2+) release.