L-Type Ca(2+) channel charge movement and intracellular Ca(2+) in skeletal muscle fibers from aging mice

In this work we tested the hypothesis that skeletal muscle fibers from aging mice exhibit a significant decline in myoplasmic Ca(2+) concentration resulting from a reduction in L-type Ca(2+) channel (dihydropyridine receptor, DHPR) charge movement. Skeletal muscle fibers from the flexor digitorum brevis (FDB) muscle were obtained from 5-7-, 14-18-, or 21-24-month-old FVB mice and 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). Total charge movement or the DHPR charge movement was measured simultaneously with intracellular Ca(2+) concentration. The maximum charge movement (Q(max)) recorded (mean +/- SEM, in nC microF(-1)) was 53 +/- 3.2 (n = 47), 51 +/- 3.2 (n = 35) (non-significant, ns), and 33 +/- 1.9 (n = 32) (p < 0.01), for the three age groups, respectively. Q(max) corresponding to the DHPR was 43 +/- 3.3, 38 +/- 4.1 (ns), and 25 +/- 3.4 (p < 0.01) for the three age groups, respectively. The peak intracellular [Ca(2+)] recorded at 40 mV (in microM) was 15.7 +/- 0. 12, 16.7 +/- 0.18 (ns), and 8.2 +/- 0.07 (p < 0.01) for the three age groups, 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 the reduction in the peak intracellular [Ca(2+)] results from a larger number of ryanodine receptors uncoupled to DHPRs in skeletal muscle fibers from aging mammals.     

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.     

Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.     

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.     

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.     

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.     

Differential impact of mitochondrial positioning on mitochondrial Ca(2+) uptake and Ca(2+) spark suppression in skeletal muscle

Muscle contraction requires ATP and Ca(2+) and, thus, is under direct control of mitochondria and the sarcoplasmic reticulum. During postnatal skeletal muscle maturation, the mitochondrial network exhibits a shift from a longitudinal ("longitudinal mitochondria") to a mostly transversal orientation as a result of a progressive increase in mitochondrial association with Ca(2+) release units (CRUs) or triads ("triadic mitochondria"). To determine the physiological implications of this shift in mitochondrial disposition, we used confocal microscopy to monitor activity-dependent changes in myoplasmic (fluo 4) and mitochondrial (rhod 2) Ca(2+) in single flexor digitorum brevis (FDB) fibers from 1- to 4-mo-old mice. A robust and sustained Ca(2+) accumulation in triadic mitochondria was triggered by repetitive tetanic stimulation (500 ms, 100 Hz, every 2.5 s) in FDB fibers from 4-mo-old mice. Specifically, mitochondrial rhod 2 fluorescence increased 272 ± 39% after a single tetanus and 412 ± 45% after five tetani and decayed slowly over 10 min following the final tetanus. Similar results were observed in fibers expressing mitochondrial pericam, a mitochondrial-targeted ratiometric Ca(2+) indicator. Interestingly, sustained mitochondrial Ca(2+) uptake following repetitive tetanic stimulation was similar for triadic and longitudinal mitochondria in FDB fibers from 1-mo-old mice, and both mitochondrial populations were found by electron microscopy to be continuous and structurally tethered to the sarcoplasmic reticulum. Conversely, the frequency of osmotic shock-induced Ca(2+) sparks per CRU density decreased threefold (from 3.6 ± 0.2 to 1.2 ± 0.1 events·CRU(-1)·min(-1)·100 μm(-2)) during postnatal development in direct linear correspondence (r(2) = 0.95) to an increase in mitochondrion-CRU pairing. Together, these results indicate that mitochondrion-CRU association promotes Ca(2+) spark suppression but does not significantly impact mitochondrial Ca(2+) uptake.     

Expression of the Na(+)/Ca(2+) exchanger in skeletal muscle

The expression of the Na(+)/Ca(2+) exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na(+)/Ca(2+) exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.     

Spatial Ca(2+) distribution in contracting skeletal and cardiac muscle cells

The spatiotemporal distribution of intracellular Ca(2+) release in contracting skeletal and cardiac muscle cells was defined using a snapshot imaging technique. Calcium imaging was performed on intact skeletal and cardiac muscle cells during contractions induced by an action potential (AP). The sarcomere length of the skeletal and cardiac cells was approximately 2 micrometer. Imaging Rhod-2 fluorescence only during a very brief (7 ns) snapshot of excitation light minimized potential image-blurring artifacts due to movement and/or diffusion. In skeletal muscle cells, the AP triggered a large fast Ca(2+) transient that peaked in less than 3 ms. Distinct subsarcomeric Ca(2+) gradients were evident during the first 4 ms of the skeletal Ca(2+) transient. In cardiac muscle, the AP-triggered Ca(2+) transient was much slower and peaked in approximately 100 ms. In contrast to the skeletal case, there were no detectable subsarcomeric Ca(2+) gradients during the cardiac Ca(2+) transient. Theoretical simulations suggest that the subsarcomeric Ca(2+) gradients seen in skeletal muscle were detectable because of the high speed and synchrony of local Ca(2+) release. Slower asynchronous recruitment of local Ca(2+) release units may account for the absence of detectable subsarcomeric Ca(2+) gradients in cardiac muscle. The speed and synchrony of local Ca(2+) gradients are quite different in AP-activated contracting cardiac and skeletal muscle cells at normal resting sarcomere lengths.     

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.     

Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.     

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.     

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.     

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.     

Inhibition of muscle carbonic anhydrase slows the Ca(2+) transient in rat skeletal muscle fibers

A countertransport ofH⁺ is coupled to Ca²⁺ transport across thesarcoplasmic reticulum (SR) membrane. We propose that SR carbonic anhydrase (CA) accelerates the CO₂-HCO reaction so that H⁺ ions, which are exchanged forCa²⁺ ions, are produced or buffered in the SR at sufficientrates. Inhibition of this SR-CA is expected to reduce the rate ofH⁺ fluxes, which then will retard the kinetics ofCa²⁺ transport. Fura 2 signals and isometric force weresimultaneously recorded in fiber bundles of the soleus (SOL) andextensor digitorum longus (EDL) from rats in the absence and presenceof the lipophilic CA inhibitors L-645151, chlorzolamide (CLZ), andethoxzolamide (ETZ), as well as the hydrophilic inhibitor acetazolamide(ACTZ). Fura 2 and force signals were analyzed for time to peak (TTP), 50% decay time (t₅₀), and their amplitudes.L-645151, CLZ, and ETZ significantly increased TTP of fura 2 by10-25 ms in SOL and by 5-7 ms in EDL and TTP of force by6-30 ms in both muscles. L-645151 and ETZ significantly prolongedt₅₀ of fura 2 and force by 20-55 and40-160 ms, respectively, in SOL and EDL. L-645151, CLZ, and ETZalso increased peak force of single twitches and amplitudes of furafluorescence ratio (R_340/380) at an excitation wavelengthof 340 to 380 nm. All effects of CA inhibitors on fura 2 and forcesignals could be reversed. ACTZ did not affect TTP, t₅₀, and amplitudes of fura 2 signals or force.L-645151, CLZ, and ETZ had no effects on myosin-, Ca²⁺-,and Na⁺-K⁺-ATPase activities, nor did theyaffect the amplitude and half-width of action potentials. We concludethat inhibition of SR-CA by impairing H⁺ countertransportis responsible for deceleration of intracellular Ca²⁺transients and contraction times.

     

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.     

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.

     

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.