Patch-clamp recording of charge movement, Ca(2+) current, and Ca(2+) transients in adult skeletal muscle fibers

Intramembrane charge movement (Q), Ca(2+) conductance (G(m)) through the dihydropyridine-sensitive L-type Ca(2+) channel (DHPR) and intracellular Ca(2+) fluorescence (F) have been recorded simultaneously in flexor digitorum brevis muscle fibers of adult mice, using the whole-cell configuration of the patch-clamp technique. The voltage distribution of Q was fitted to a Boltzmann equation; the Q(max), V(1/2Q), and effective valence (z(Q)) values were 41 +/- 3.1 nC/&mgr;F, -17.6 +/- 0.7 mV, and 2.0 +/- 0.12, respectively. V(1/2G) and z(G) values were -0.3 +/- 0.06 mV and 5.6 +/- 0.34, respectively. Peak Ca(2+) transients did not change significantly after 30 min of recording. F was fit to a Boltzmann equation, and the values for V(F1/2) and z(F) were 6.2 +/- 0.04 mV and 2.4, respectively. F was adequately fit to the fourth power of Q. These results demonstrate that the patch-clamp technique is appropriate for recording Q, G(m), and intracellular [Ca(2+)] simultaneously in mature skeletal muscle fibers and that the voltage distribution of the changes in intracellular Ca(2+) can be predicted by a Hodgkin-Huxley model.     

Sustained overexpression of IGF-1 prevents age-dependent decrease in charge movement and intracellular Ca(2+) in mouse skeletal muscle

In this work we tested the hypothesis that transgenic sustained overexpression of IGF-1 prevents age-dependent decreases in charge movement and intracellular Ca(2+) in skeletal muscle fibers. To this end, short flexor digitorum brevis (FDB) muscle fibers from 5-7- and 21-24-month-old FVB (wild-type) and S1S2 (IGF-1 transgenic) mice were studied. Fibers were voltage-clamped in the whole-cell configuration of the patch-clamp technique according to described procedures (Wang, Z. M., M. L. Messi, and O. Delbono. 1999. Biophys. J. 77:2709-2716). Charge movement and intracellular Ca(2+) concentration were recorded simultaneously. The maximum charge movement (Q(max)) recorded in young wild-type and transgenic mice was (mean +/- SEM, in nC microF(-1)): 52 +/- 2.1 (n = 46) and 54 +/- 1.9 (n = 38) (non-significant, ns), respectively, whereas in old wild-type and old transgenic mice the values were 36 +/- 2.1 (n = 32) and 49 +/- 2.3 (n = 35), respectively (p < 0.01). The peak intracellular calcium [Ca(2+)](i) recorded in young wild-type and transgenic mice was (in muM): 14.5 +/- 0.9 and 16 +/- 2.1 (ns), whereas in old wild-type and transgenic mice the values were 9.9 +/- 0.1 and 14 +/- 1.1 (p < 0.01), respectively. No significant changes in the voltage distribution or steepness of the Q-V or [Ca(2+)]-V relationship were found. These data support the concept that overexpression of IGF-1 in skeletal muscle prevents age-dependent reduction in charge movement and peak [Ca(2+)](i).     

Ca(2+) influx and opening of Ca(2+)-activated K(+) channels in muscle fibers from control and mdx mice

Using the patch-clamp technique, we demonstrate that, in depolarized cell-attached patches from mouse skeletal muscle fibers, a short hyperpolarization to resting value is followed by a transient activation of Ca(2+)-activated K(+) channels (K(Ca)) upon return to depolarized levels. These results indicate that sparse sites of passive Ca(2+) influx at resting potentials are responsible for a subsarcolemmal Ca(2+) load high enough to induce K(Ca) channel activation upon muscle activation. We then investigate this phenomenon in mdx dystrophin-deficient muscle fibers, in which an elevated Ca(2+) influx and a subsequent subsarcolemmal Ca(2+) overload are suspected. The number of Ca(2+) entry sites detected with K(Ca) was found to be greater in mdx muscle. K(Ca) activity reflecting subsarcolemmal Ca(2+) load was also found to be independent of the activity of leak channels carrying inward currents at negative potentials in mdx muscle. These results indicate that the sites of passive Ca(2+) influx newly described in this study could represent the Ca(2+) influx pathways responsible for the subsarcolemmal Ca(2+) overload in mdx muscle fibers.     

Intramembrane charge movement and L-type calcium current in skeletal muscle fibers isolated from control and mdx mice

Dystrophin-deficient muscle fibers from mdx mice are believed to suffer from increased calcium entry and elevated submembranous calcium level, the actual source and functional consequences of which remain obscure. Here we compare the properties of the dihydropyridine receptor as voltage sensor and calcium channel in control and mdx muscle fibers, using the silicone-voltage clamp technique. In control fibers charge movement followed a two-state Boltzmann distribution with values for maximal charge, midpoint voltage, and steepness of 23 +/- 2 nC/ micro F, -37 +/- 3 mV, and 13 +/- 1 mV (n = 7). Essentially identical values were obtained in mdx fibers and the time course of charge recovery from inactivation was also similar in the two populations (tau approximately 6 s). In control fibers the voltage dependence of the slow calcium current elicited by 100-ms-long pulses gave values for maximal conductance, apparent reversal potential, half-activation potential, and steepness factor of 156 +/- 15 S/F, 65.5 +/- 2.9 mV, -0.76 +/- 1.2 mV, and 6.2 +/- 0.5 mV (n = 17). In mdx fibers, the half-activation potential of the calcium current was slightly more negative (-6.2 +/- 1.2 mV, n = 16). Also, when using longer pulses, the time constant of calcium current decay was found to be significantly larger (by a factor of 1.5-2) in mdx than in control fibers. These changes in calcium current properties are unlikely to be primarily responsible for a dramatic alteration of intracellular calcium homeostasis. They may be speculated to result, at least in part, from remodeling of the submembranous cytoskeleton network due to the absence of dystrophin.     

Sarcoplasmic reticulum Ca2+ release declines in muscle fibers from aging mice

This study hypothesized that decline in sarcoplasmic reticulum (SR) Ca²⁺ release and maximal SR-releasable Ca²⁺ contributes to decreased specific force with aging. To test it, we recorded electrically evoked maximal isometric specific force followed by 4-chloro-m-cresol (4-CmC)-evoked maximal contracture force in single intact fibers from the mouse flexor digitorum brevis muscle. Significant differences in tetanic, but not in 4-CmC-evoked, contracture forces were recorded in fibers from aging mice as compared to younger mice. Peak intracellular Ca²⁺ in response to 4-CmC did not differ significantly. SR Ca²⁺ release was recorded in whole-cell patch-clamped fibers in the linescan mode of confocal microscopy using a low-affinity Ca²⁺ indicator (Oregon green bapta-5N) with high-intracellular ethylene glycol-bis(α-aminoethyl ether)-N,N,N′N′-tetraacetic acid (20 mM). Maximal SR Ca²⁺ release, but not voltage dependence, was significantly changed in fibers from old compared to young mice. Increasing the duration of fiber depolarization did not increase the maximal rate of SR Ca²⁺ release in fibers from old compared to young mice. Voltage-dependent inactivation of SR Ca²⁺ release did not differ significantly between fibers from young and old mice. These findings indicate that alterations in excitation-contraction coupling, but not in maximal SR-releasable Ca²⁺, account for the age-dependent decline in intracellular Ca²⁺ mobilization and specific force.     

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.     

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.     

Evidence for a role of C-terminus in Ca(2+) inactivation of skeletal muscle Ca(2+) release channel (ryanodine receptor).

Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca(2+) release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca(2+) dependence in [(3)H]ryanodine binding and single channel measurements. The results indicate that the C-terminal one-fourth has a major role in Ca(2+) activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [(3)H]ryanodine binding affinity while maintaining channel activity.     

Effects of quercetin on single Ca(2+) release channel behavior of skeletal muscle

Quercetin, a bioflavonoid, is known to affect Ca(2+) fluxes in sarcoplasmic reticulum, although its direct effect on Ca(2+) release channel (CRC) in sarcoplasmic reticulum has remained to be elucidated. The present study examined the effect of quercetin on the behavior of single skeletal CRC in planar lipid bilayer. The effect of caffeine was also studied for comparison. At very low [Ca(2+)](cis) (80 pM), quercetin activated CRC marginally, whereas at elevated [Ca(2+)](cis) (10 microM), both open probability (P(o)) and sensitivity to the drug increased markedly. Caffeine showed a similar tendency. Analysis of lifetimes for single CRC showed that quercetin and caffeine led to different mean open-time and closed-time constants and their proportions. Addition of 10 microM ryanodine to CRC activated by quercetin or caffeine led to the typical subconductance state (approximately 54%) and a subsequent addition of 5 microM ruthenium red completely blocked CRC activity. When 6 microM quercetin and 3 mM caffeine were added together to the cis side of CRC, a time-dependent increase of P(o) was observed (from mode 1 (0.376 +/- 0.043, n = 5) to mode 2 (0.854 +/- 0.062, n = 5)). On the other hand, no further activation was observed when quercetin was added after caffeine. Quercetin affected only the ascending phase of the bell-shaped Ca(2+) activation/inactivation curve, whereas caffeine affected both ascending and descending phases. [(3)H]ryanodine binding to sarcoplasmic reticulum showed that channel activity increased more by both quercetin and caffeine than by caffeine alone. These characteristic differences in the modes of activation of CRC by quercetin and caffeine suggest that the channel activation mechanisms and presumably the binding sites on CRC are different for the two drugs.     

Asymmetric arrangement of auxiliary subunits of skeletal muscle voltage-gated l-type Ca(2+) channel

Highly purified L-type Ca(2+) channel complexes containing all five subunits (alpha(1), alpha(2), beta, gamma, and delta) and complexes of alpha(1)-beta subunits were obtained from skeletal muscle triad membranes by three-step purification and by 1% Triton X-100 treatment, respectively. Their structures and the subunit arrangements were analyzed by electron microscopy. Projection images of negatively stained Ca(2+) channels and alpha(1)-beta complexes were aligned, classified and averaged. The alpha(1)-beta complex showed a hollow trapezoid shape of 12 nm height. In top view, four asymmetric domains surrounded a central depression predicted to form the channel pore. The complete Ca(2+) channel complex exhibited the cylindrical shape of 20 nm in height binding a spherical domain on one edge. Further image analysis of higher complexes of the Ca(2+) channel using a monoclonal antibody against the beta subunit showed that the alpha(1)-beta complex forms the non-decorated side of the cylinder, which can traverse the membrane from outside the cell to the cytoplasm. Based on these results, we propose that the Ca(2+) channel exhibits an asymmetric arrangement of auxiliary subunits.     

Ryanodine interferes with charge movement repriming in amphibian skeletal muscle fibers.

Cut twitch muscle fibers mounted in a triple Vaseline-gap chamber were used to study the effects of ryanodine on intramembranous charge movement, and in particular on the repriming of charge 1. Charge 1 repriming was measured either under steady-state conditions or by using a pulse protocol designed to study the time course of repriming. This protocol consisted of repolarizing the fibers to -100 mV from a holding potential of 0 mV, and then measuring the reprimed charge moving in the potential range between -40 and +20 mV. Ryanodine at a high concentration (100 microM) did not affect the maximum amount of movable charge 1 and charge 2, or their voltage dependence. This indicates that the alkaloid does not interact with the voltage sensor molecules. However, ryanodine did reduce the amount of reprimed charge 1 by approximately 60% suggesting the possibility of a retrograde interaction between ryanodine receptors and voltage sensors.     

Ca(2+)-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET

To obtain information on Ca(2+)-induced tropomyosin (Tm) movement in Ca(2+)-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DD) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca(2+) causes azimuthal movement of Tm of the magnitude suggested by structural data (C. Xu, R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Biophys. J. 77:985-992). However, Ca(2+) produced a small but significant change in our phase/modulation versus frequency data, showing that changes in lifetime decay can be detected even when a change of the steady-state transfer efficiency is very small. A change in Tm azimuthal position of 17 on the actin filament obtained with the AC model indicates that solution data are in reasonable agreement with EM image reconstruction data. In addition, the data indicate that Tm also appears to rotate about its axis, resulting in a rolling motion over the F-actin surface. The DD model showed that the distance from one of the two chains of Tm to F-actin was mainly affected, further verifying that Ca(2+) causes Tm to roll over the F-actin surface. The width of the distance distributions indicated that the position of Tm in absence and in presence of Ca(2+) is well defined with appreciable local flexibility.     

Ca(2+)-channels and adrenoceptors in diabetic skeletal muscle.

The status of Ca(2+)-channels and adrenoceptors in the hind leg skeletal muscle was examined in rats 8 weeks after inducing diabetes by an intravenous injection of streptozotocin (65 mg/kg). Scatchard plot analysis of the data on specific binding of 3H-nitrendipine with crude membranes from diabetic muscle revealed an increase in the density of Ca(2+)-channels without any significant change in their affinity for the ligand. An increase in the density of beta-adrenoceptors without any alteration in their affinity, as measured by 3H-dihydroalprenolol binding, was also evident in the diabetic muscle. The observed increase in the number of Ca2+ channels or beta-adrenoceptors seems specific since no change in the alpha-adrenoceptor density or affinity, as measured by 3H-prazosin binding, was seen in the diabetic membranes. These results support the view that higher activities of Ca2+ transport systems or regulatory mechanisms may be associated with hyperfunction of the diabetic skeletal muscle.     

Functional impact of the ryanodine receptor on the skeletal muscle L-type Ca(2+) channel

L-type Ca(2+) channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H. T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:72-75.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and "RyR1-knockout" (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Q(max), 7.5 +/- 0.8 and 4.5 +/- 0.4 nC/muF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (G(max)/Q(max)). Thus, RyR1 enhances both the expression level and Ca(2+) conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (A(fast) and A(slow)) and time constants (tau(slow) and tau(fast)) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; A(fast)/[A(fast) + A(slow)] approximately 0.25). However, both tau(fast) and tau(slow) were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (tau(fast), 8.5 +/- 1.2 and 4.4 +/- 0.5 ms; tau(slow), 79.5 +/- 10.5 and 34.6 +/- 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (-) Bay K 8644 (5 microM) caused a hyperpolarizing shift (approximately 10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both A(slow) and A(fast) in normal myotubes, but a large increase in only A(fast) in dyspedic myotubes. Equimolar substitution of Ba(2+) for extracellular Ca(2+) increased both A(fast) and A(slow) in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either A(fast) or A(slow). These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca(2+) channel accessory subunit.     

Ca(2+) sensors of L-type Ca(2+) channel

Ca(2+)-induced inactivation of L-type Ca(2+) is differentially mediated by two C-terminal motifs of the alpha(1C) subunit, L (1572-1587) and K (1599-1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca(2+) sensor and an adjacent Ca(2+)-independent tethering site for calmodulin. The Ca(2+) sensor contributes to higher Ca(2+) sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca(2+)-dependent current decay, the two-site modulation by Ca(2+) and calmodulin may underlie Ca(2+)-induced inactivation of the channel.     

Mutation of divergent region 1 alters caffeine and Ca(2+) sensitivity of the skeletal muscle Ca(2+) release channel (ryanodine receptor)

Replacement of amino acids 4187-4628 in the skeletal muscle Ca(2+) release channel (skeletal ryanodine receptor (RyR1)), including nearly all of divergent region 1 (amino acids 4254-4631), with the corresponding cardiac ryanodine receptor (RyR2) sequence leads to increased sensitivity of channel activation by caffeine and Ca(2+) and to decreased sensitivity of channel inactivation by elevated Ca(2+) (Du, G. G., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 26120-26126). In further investigations, this region was subdivided by the construction of new chimeras, and alterations in channel function were detected by measurement of the caffeine dependence of in vivo Ca(2+) release and the Ca(2+) dependence of [(3)H]ryanodine binding. Chimera RF10a (amino acids 4187-4381) had a lower EC(50) value for activation by caffeine, and RF10c (4557-4628) had a higher EC(50) value, whereas the EC(50) value for chimera RF10b (4382-4556) was unchanged. Chimeras RF10b and RF10c were more sensitive to activation by Ca(2+), whereas RF10a was less sensitive to inactivation by Ca(2+), implicating RF10b and RF10c in Ca(2+) activation and RF10a in Ca(2+) inactivation. Deletion of much of divergent region 1 sequence to create mutant Delta4274-4535 led to higher caffeine and Ca(2+) sensitivity of channel activation and to lower Ca(2+) sensitivity for inactivation. Thus, deletion results demonstrate that caffeine, Ca(2+), and ryanodine binding sites are not located in amino acids 4274-4535. Nevertheless, the properties of the deletion and chimeric mutants demonstrate that amino acids 4274-4535 and three shorter sequences in this region (F10a, amino acids 4187-4381; F10b, 4382-4556; and F10c, 4557-4628) in RyR1 modulate Ca(2+) and caffeine sensitivity of the Ca(2+) release channel.     

Characterization of intracellular Ca(2+) stores in gallbladder smooth muscle

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.     

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.     

Triadins modulate intracellular Ca(2+) homeostasis but are not essential for excitation-contraction coupling in skeletal muscle

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca(2+) imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca(2+) transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca(2+).[(3)H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca(2+) activation or Ca(2+) and Mg(2+) inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca(2+) homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.     

Adenosine(5')hexaphospho(5')adenosine stimulation of a Ca(2+)-induced Ca(2+)-release channel from skeletal muscle sarcoplasmic reticulum.

Stimulation of a Ca(2+)-induced Ca(2+)-release channel from skeletal muscle sarcoplasmic reticulum by various adenosine(5')oligophospho(5')adenosines (ApnA, n = 2-6) by a rapid quenching technique using radioactive calcium was studied. Ap4A, Ap5A and Ap6A, as well as adenosine 5'-[beta, gamma-methylene]triphosphate (AdoPP [CH2]P), a non-hydrolyzable ATP analogue, stimulated the Ca(2+)-release channel, whereas Ap2A and Ap3A had no effect. At a concentration of 0.5 mM, the order of stimulation was AdoPP[CH2]P less than Ap4A less than Ap5A much less than Ap6A. As well as having the highest affinity (0.44 mM for half-maximal stimulation), Ap6A showed an extraordinarily high Hill coefficient of 3.3 (1.9 for AdoPP[CH2]P, 2.1 for Ap5A). The stimulating effect of Ap6A was reversible, yet its dissociation proceeded very slowly. Stimulation of Ca2+ release by Ap6A was counteracted by Mg2+ and ruthenium red. A 2',3'-dialdehyde derivative of Ap6A, which is a chemical probe for amino groups, stimulated irreversibly the Ca(2+)-release channel and modified some high-molecular-mass sarcoplasmic reticulum proteins, possibly including the channel protein. Our data suggest that Ap6A stimulates the Ca2+ channel by binding to the activation site of the channel subunit and simultaneously preventing the spontaneous decay of the Ca2+ channel by keeping together two of the four channel subunits by bridging them with its two adenosine groups.