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

In skeletal muscle fibers, the intracellular loop between domains II and III of the alpha(1)-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca(2+) release channel in the sarcoplasmic reticulum. We examined the effects of synthetic peptide segments of this loop on Ca(2+) release in mechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg(2+) concentration ([Mg(2+)]; 1 mM), a 20-residue skeletal muscle DHPR peptide [A(S(20)); Thr(671)-Leu(690); 30 microM], shown previously to induce Ca(2+) release in a triad preparation, caused only small spontaneous force responses in approximately 40% of fibers, although it potentiated responses to depolarization and caffeine in all fibers. The COOH-terminal half of A(S(20)) [A(S(10))] induced much larger spontaneous responses but also caused substantial inhibition of Ca(2+) release to both depolarization and caffeine. Both peptides induced or potentiated Ca(2+) release even when the voltage sensors were inactivated, indicating direct action on the Ca(2+) release channels. The corresponding 20-residue cardiac DHPR peptide [A(C(20)); Thr(793)-Ala(812)] was ineffective, but its COOH-terminal half [A(C(10))] had effects similar to A(S(20)). In the presence of lower [Mg(2+)] (0.2 mM), exposure to either A(S(20)) or A(C(10)) (30 microM) induced substantial Ca(2+) release. Peptide C(S) (100 microM), a loop segment reported to inhibit Ca(2+) release in triads, caused partial inhibition of depolarization-induced Ca(2+) release. In toad fibers, each of the A peptides had effects similar to or greater than those in rat fibers. These findings suggest that the A and C regions of the skeletal DHPR II-III loop may have important roles in vivo.

Postulated role of interdomain interaction within the ryanodine receptor in Ca(2+) channel regulation.

Localized distribution of malignant hyperthermia (MH) and central core disease (CCD) mutations in N-terminal and central domains of the ryanodine receptor suggests that the interaction between these domains may be involved in Ca(2+) channel regulation. To test this hypothesis, we investigated the effects of a new synthetic domain peptide DP4 corresponding to the Leu(2442)-Pro(2477) region of the central domain. DP4 enhanced ryanodine binding and induced a rapid Ca(2+) release. The concentration for half-maximal activation by agonists was considerably reduced in the presence of DP4. These effects of DP4 are analogous to the functional modifications of the ryanodine receptor caused by MH/CCD mutations (viz. hyperactivation of the channel and hypersensitization of the channel to agonists). Replacement of Arg of DP4 with Cys, mimicking the in vivo Arg(2458)-to-Cys(2458) mutation, abolished the activating effects of DP4. An N-terminal domain peptide DP1 (El-Hayek, R., Saiki, Y., Yamamoto, T., and Ikemoto, N. (1999) J. Biol. Chem. 274, 33341-33347) shows similar activation/sensitization effects. The addition of both DP4 and DP1 produced mutual interference of their activating functions. We tentatively propose that contact between the two (N-terminal and central) domains closes the channel, whereas removal of the contact by these domain peptides or by MH/CCD mutations de-blocks the channel, resulting in hyperactivation/hyper-sensitization effects.

A postulated role of the near amino-terminal domain of the ryanodine receptor in the regulation of the sarcoplasmic reticulum Ca(2+) channel.

To test the hypothesis that interactions among several putative domains of the ryanodine receptor (RyR) are involved in the regulation of its Ca(2+) release channel, we synthesized several peptides corresponding to selected NH(2)-terminal regions of the RyR. We then examined their effects on ryanodine binding and Ca(2+) release activities of the sarcoplasmic reticulum isolated from skeletal and cardiac muscle. Peptides 1-2s, 1-2c, and 1 enhanced ryanodine binding to cardiac RyR and induced a rapid Ca(2+) release from cardiac SR in a dose-dependent manner. The order of the potency for the activation of the Ca(2+) release channel was 1-2c > 1 > 1-2s. Interestingly, these peptides produced significant activation of the cardiac RyR at near zero or subactivating [Ca(2+)], indicating that the peptides enhanced the Ca(2+) sensitivity of the channel. Peptides 1-2c, 1-2s, and 1 had virtually no effect on skeletal RyR, although occasional and variable extents of activation were observed in ryanodine binding assays performed at 36 degrees C. Peptide 3 affected neither cardiac nor skeletal RyR. We propose that domains 1 and 1-2 of the RyR, to which these activating peptides correspond, would interact with one or more other domains within the RyR (including presumably the Ca(2+)-binding domain) to regulate the Ca(2+) channel.

Involvement of the Glu724-Pro760 region of the dihydropyridine receptor II-III loop in skeletal muscle-type excitation-contraction coupling.

Our previous study (El-Hayek, R., Antoniu, B., Wang, J. P., Hamilton, S. L., and Ikemoto, N. (1995) J. Biol. Chem. 270, 22116-22118) suggested the hypothesis that skeletal muscle-type excitation-contraction coupling is regulated by two domains (activating and blocking) of the II-III loop of the dihydropyridine receptor alpha1 subunit. We investigated this hypothesis by examining conformational changes in the ryanodine receptor induced by synthetic peptides and by transverse tubular system (T-tubule) depolarization. Peptide A, corresponding to the Thr671-Leu690 region, rapidly changed the ryanodine receptor conformation from a blocked state (low fluorescence of the conformational probe, methyl coumarin acetamide, attached specifically to the ryanodine receptor) to an activated state (high methyl coumarin acetamide fluorescence) as T-tubule depolarization did. Peptide C, corresponding to the Glu724-Pro760 region, blocked both conformational changes induced by peptide A and T-tubule depolarization. Its ability to block peptide A-induced and depolarization-induced activation was considerably impaired by replacing the portion of peptide C corresponding to the Phe725-Pro742 region of the loop with cardiac muscle-type sequence. These results are consistent with the model that depolarization-induced activation of excitation-contraction coupling and blocking/repriming are mediated by the peptide A region and the peptide C region (containing the critical Phe725-Pro742 sequence) of the II-III loop, respectively.

Immunophenotypic pattern of childhood acute lymphoblastic leukemia.

Full text is available as a scanned copy of the original print version.

Identification of the minimum essential region in the II-III loop of the dihydropyridine receptor alpha 1 subunit required for activation of skeletal muscle-type excitation-contraction coupling.

We have previously shown that among several peptides encompassing various regions of the II-III loop of the dihydropyridine receptor alpha 1 subunit, only one peptide corresponding to the Thr671-Leu690 region (designated as peptide A) activated ryanodine binding to and induced calcium release from the sarcoplasmic reticulum [El-Hayek et al. (1995) J. Biol. Chem. 270, 22116-22118]. To further localize within peptide A the minimum unit essential for activating the sarcoplasmic reticulum calcium release channel, we synthesized variously truncated forms of peptide A and examined their ability to activate ryanodine binding. We found that the carboxy-terminal 10-residue region of peptide A encompassing Arg681-Leu690 (peptide As-10; s, skeletal muscle-type sequence) activated ryanodine binding in a RyR1-specific manner and induced calcium release even more efficiently than the 20-residue peptide A. Further truncation of one or more residue(s) of peptide As-10 virtually abolished both functions of activating ryanodine binding and inducing Ca2+ release. The activating ability of As-10 seems to be determined by at least two factors: (1) the distribution of the positively charged residues, and (2) the skeletal muscle-type amino acid sequence, as deduced from the comparison of various peptides with modified structures. These results provide evidence that the minimum essential unit for the in situ trigger of skeletal muscle excitation-contraction coupling is localized in the Arg681-Leu690 region of the II-III loop of the alpha 1 subunit of the dihydropyridine receptor.

Signal transmission and transduction in excitation-contraction coupling.

For the better understanding of the molecular mechanism of E-C coupling, two key questions remain to be resolved: (a) how the excitation signal elicited in the T-tubule membrane is transmitted to the ryanodine receptor, RyR (signal transmission), and (b) how the signal transmitted from the T-tubule to the RyR is translated into the action of opening the sarcoplasmic reticulum Ca2+ channel to induce Ca2+ release and muscle contraction (signal transduction). Our recent studies on E-C coupling with the use of the isolated triads and synthetic peptides have provided several pieces of new information. It appears that the signal transmission is mediated by the voltage-controlled binding of the Thr671-Leu690 region (Trigger) of the cytoplasmic II-III loop of the dihydropyridine receptor alpha 1 a subunit to the putative activator site on the RyR. The transmitted signal is translated to the action of channel opening by mediation of rapid conformational changes occurring in the RyR. Upon T-tubule polarization the Glu724-Pro760 region of the loop (Blocker) replaces the RyR-bound Trigger. This reprimes the RyR to the original conformational state.

Reciprocal control of the conformational state of the sarcoplasmic reticulum calcium channel protein by polarization and depolarization in the transverse tubule.

We attached the conformational probe methylcoumarin acetate (MCA) specifically to the junctional foot protein (JFP) moiety of triads, and monitored conformational changes in the JFP during polarization and depolarization of the T-tubule moiety. The MCA fluorescence decreased upon T-tubule polarization, and the fluorescence changes were blocked by preventing T-tubule polarization or by a nimodipine block of the T-tubule-to-sarcoplasmic reticulum communication. Depolarization of the T-tubule reversed the MCA fluorescence decrease which had been produced by T-tubule polarization. These results suggest that the conformational and functional states of the JFP are regulated by T-tubule polarization and depolarization in a reciprocal fashion.

Peptide probe of ryanodine receptor function. Imperatoxin A, a peptide from the venom of the scorpion Pandinus imperator, selectively activates skeletal-type ryanodine receptor isoforms.

We have used [3H]ryanodine binding experiments and single channel recordings to provide convergent descriptions of the effect of imperatoxin A (IpTxa), a approximately 5-kDa peptide from the venom of the scorpion Pandinus imperator (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Ntl. Acad. Sc. U.S.A. 89, 12185-12189) on Ca2+ release channels/ryanodine receptors (RyR) of sarcoplasmic reticulum (SR). At nanomolar concentrations, IpTxa increased the binding of [3H]ryanodine to skeletal SR and, to a lesser extent, to cerebellum microsomes. The activating effect of IpTxa on skeletal SR was Ca(2+)-dependent, synergized by caffeine, and independent of other modulators of RyRs. However, IpTxa had negligible effects on tissues where the expression of skeletal-type RyR isoforms (RyR1) is small or altogether absent, i.e. cardiac, cerebrum, and liver microsomes. Thus, IpTxa may be used as a ligand capable of discriminating between RyR isoforms with nanomolar affinity. IpTxa increased the open probability (Po) of rabbit skeletal muscle RyRs by increasing the frequency of open events and decreasing the duration of the closed lifetimes. This activating effect was dose-dependent (ED50 = 10 nM), had a fast onset, and was fully reversible. Purified RyR from solubilized skeletal SR displayed high affinity for [3H]ryanodine with a KD of 6.1 nM and Bmax of approximately 30 pmol/mg of protein. IpTxa increased [3H]ryanodine binding noncompetitively by increasing Bmax to approximately 60 pmol/mg of protein. These results suggested the presence of an IpTxa-binding site on the RyR or a closely associated regulatory protein. This site appears to be distinct from the caffeine- and adenine nucleotide-regulatory sites. IpTxa may prove a useful tool to identify regulatory domains critical for channel gating and to dissect the contribution of skeletal-type RyRs to intracellular Ca2+ waveforms generated by stimulation of different RyR isoforms.

Effects of perchlorate on depolarization-induced conformational changes in the junctional foot protein and Ca2+ release from sarcoplasmic reticulum.

Perchlorate is one of the most potent activators of skeletal muscle excitation-contraction (E-C) coupling reported in the literature, but the detailed mechanism of its action remains to be elucidated. In an attempt to further resolve the mode of perchlorate action, the effects of increasing concentrations of perchlorate on the voltage-dependent (T-tubule-mediated) and voltage-independent portions of Ca2+ release were investigated using the isolated triad model. Low concentrations of perchlorate (< or = 10 mM) activated SR Ca2+ release only when the T-tubule moiety was chemically depolarized. Higher concentrations of perchlorate (30-100 mM), on the other hand, produced significant activation of SR Ca2+ release, regardless of whether or not the T-tubule was depolarized. In order to gain further insights, we monitored the conformational change in the junctional foot protein (JFP), which presumably is an important intermediate step in E-C coupling [Yano, M., El-Hayek, R., & Ikemoto, N. (1995) J. Biol. Chem. 270, 3017-3021], using the fluorescently labeled triad preparation. Again, low concentrations of perchlorate (< or = 10 mM) produced a preferential activation of voltage-dependent protein conformational change, while higher concentrations of perchlorate produced significant activation of voltage-independent protein conformational change. An increase in the ryanodine binding by perchlorate occurred only in the higher concentration range where the voltage-independent protein conformational change was activated. These results suggest that perchlorate activates E-C coupling by acting on at least two different steps: at lower concentrations, on the T-tubule-to-JFP signal transmission step; at higher concentrations, on the JFP directly.

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

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

Insulin doses in children using conventional therapy for insulin dependent diabetes.

The objective of this project was to develop plots of daily insulin dosages by percentile in diabetic children and to analyze various factors, such as metabolic control, age, and duration of diabetes that might influence the insulin prescription. Patients in extremely poor metabolic control (HbA1C > 9.8%) and patients with less than 1 year of diabetes were excluded from analysis. Patients were aged 2-21 years and the mean age at diagnosis was 8 years. Thirty-two percent of the patients were younger than 5 years and 15% were older than 15 years at diagnosis. Ninety-nine percent of patients used both regular and intermediate-acting (NPH or Lente) insulin 10-30 min before breakfast and again in the evening. Ten percent of the patients used only regular insulin at supper but used intermediate-acting insulin at bedtime. Insulin doses varied between 0.3 and 1.2 U/kg/day in prepubertal children (mean, 0.8 U/kg/day) and 0.5 and 1.8 U/kg body weight/day in pubertal children (mean, 1.25 U/kg/day). Average insulin doses in boys and girls were similar, except at ages 10-13, when the dose was significantly higher in girls. The insulin dose declined after age 17 in both genders to a value at age 21 that was 20-30% lower than the highest dose during pubertal years. Insulin dose did not correlate with duration of disease after 2 years, the ratio of morning to evening insulin, the ratio of regular to intermediate-acting insulin, or body mass. There was a slight association between higher insulin doses and higher glycosylated hemoglobin values. The results indicate that insulin requirements vary over a wide range in a group of children with metabolic control ranging from 'excellent' to 'fair' and correlate primarily with age and pubertal development. About 25% of prepubertal children and 50% of pubertal and post-pubertal children use more than 1 U insulin/kg/day--a dose commonly believed to be an 'upper limit' by many health professionals. The data should provide guidelines for the range of insulin dosing required to achieve fairly good to excellent control in diabetic children of various ages.

Role of calcium feedback in excitation-contraction coupling in isolated triads.

There is a considerable controversy in the literature concerning the effects of higher concentrations of calcium chelators (e.g. BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) or fura-2) on the intracellular Ca2+ transients in muscle. We induced calcium release from sarcoplasmic reticulum (SR) in the triad preparation by chemical depolarization of the T-tubule in the presence of various concentrations of BAPTA-calcium buffer ([Ca2+] = 0.1 microM) and investigated the effects of the BAPTA concentration on the time courses of conformational changes in the junctional foot protein (JFP) and calcium release from SR. Upon stimulation, the JFP underwent biphasic conformational changes, as determined by stopped-flow fluorometry of the JFP-bound conformational probe. The first phase of protein conformational change, which preceded calcium release from SR, was virtually unaffected by the BAPTA concentration. However, the magnitude of the second phase increased in an inversely proportional fashion to the BAPTA concentration. An abrupt increase in [Ca2+] from 0.1 microM up to 1.0 microM (delta Ca2+), concurrently with T-tubule depolarization, produced biphasic protein conformational changes: a delta Ca(2+)-independent first phase and a delta Ca(2+)-dependent second phase. Similar Ca2+ jump experiments under non-depolarizing conditions produced a slow monophasic conformational change equivalent to the second phase described above. These results suggest that the first phase of protein conformational change represents the activation of JFP by T-tubule depolarization to induce calcium release, and the second phase the secondary activation by the released Ca2+. Activation of the JFP by the released Ca2+ resulted in an acceleration of both (i) the rate of initial calcium release, and (ii) the subsequent attenuation of calcium release. The acceleration of both was suppressed by higher concentrations of BAPTA. These results provide a reasonable explanation for both of the apparently contradictory views in the literature; high concentrations of calcium buffer (a) suppress the initial activation and (b) prevent the subsequent attenuation of calcium release.

A conformational change in the junctional foot protein is involved in the regulation of Ca2+ release from sarcoplasmic reticulum. Studies on polylysine-induced Ca2+ release.

We investigated both conformational changes in the junctional foot protein (JFP) and Ca2+ release from sarcoplasmic reticulum (SR) in parallel after stimulation of triadic vesicles by the JFP-specific ligand, polylysine. To monitor protein conformational change, the JFP was labeled in a site-directed fashion with the fluorescent conformational probe methylcoumarin acetate (MCA) (Kang, J. J., Tarcsafalvi, A., Carlos, A. D., Fujimoto, E., Shahrokh, Z., Thevenin, B. J.-M., Shohet, S. B., and Ikemoto, N. (1992) Biochemistry 31, 3288-3293). The induction of SR Ca2+ release by polylysine produced a rapid increase in the fluorescence intensity of the JFP-bound MCA. The polylysine concentration dependence of the fluorescence change was essentially the same as that of Ca2+ release, suggesting that the two events are tightly coupled. However, the rate constant of MCA fluorescence change was much larger than that of Ca2+ release; i.e. the conformational change preceded Ca2+ release. Prevention of protein conformational change by lysine (0.2 M) inhibited Ca2+ release from SR. Inhibition of Ca2+ release by Mg2+ (5 mM), however, had little effect on the conformational change. These results suggest that binding of polylysine to the JFP produces conformational changes in the protein, which in turn activates the Ca2+ channel, leading to Ca2+ release from the SR.

Altered E-C coupling in triads isolated from malignant hyperthermia-susceptible porcine muscle.

Triad vesicles were isolated from normal (N) and homozygous malignant hyperthermia-susceptible (MHS) porcine skeletal muscle, and two types of sarcoplasmic reticulum Ca2+ release were investigated: 1) polylysine-induced Ca2+ release (direct stimulation of the junctional foot protein), and 2) depolarization-induced Ca2+ release (stimulation of the junctional foot protein via the dihydropyridine receptor). At submaximal concentrations of polylysine, the rates of induced Ca2+ release from the MHS triads were greater than from normal triads. The T tubules of polarized triads were depolarized by the K(+)-to-Na+ ionic replacement protocol. Higher grades of T-tubule depolarization resulted in higher rates of Ca2+ release from both MHS and normal triads but, when compared at a given grade of T-tubule depolarization, the release rate was always greater from the MHS than from normal triads. Thus the activity of the SR Ca2+ release channel is always higher in MHS than in normal muscle at a given submaximal dose of release trigger. This difference is observed when the channel is stimulated directly by polylysine or indirectly via a depolarization-induced activation of the T-tubule dihydropyridine receptor.

Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors.

Helothermine, a protein from the venom of the Mexican beaded lizard (Heloderma horridum horridum), was found to inhibit [3H]ryanodine binding to cardiac and skeletal sarcoplasmic reticulum, to block cardiac and skeletal ryanodine receptor channels incorporated into planar bilayers, and to block Ca(2+)-induced Ca2+ release triggered by photolysis of nitr-5 in saponin-permeabilized trabeculae from rat ventricle. Cloning of the helothermine cDNA revealed that the protein is composed of 223 amino acids with a molecular mass of 25,376 daltons, and apparently is stabilized by eight disulfide bridges. The peptide sequence showed significant homology with a family of cysteine-rich secretory proteins found in the male genital tract and in salivary glands. The interaction of helothermine and ryanodine receptors should serve to define functional domains within the channel structure involved in the control of Ca2+ release from sarcoplasmic reticulum.

Conformational changes in the junctional foot protein/Ca2+ release channel mediate depolarization-induced Ca2+ release from sarcoplasmic reticulum.

In an attempt to monitor the kinetic events occurring in the junctional foot protein (JFP) during excitation-contraction coupling, the JFP moiety of isolated triads was covalently labeled in a site-directed manner with methylcoumarin acetate (MCA) using a recently developed technique (Kang, J.J., Tarcsafalvi, A., Carlos, A.D., Fujimoto, E., Shahrokh, Z., Thevenin, B.J.M., Shohet, S.B., and Ikemoto, N. (1992) Biochemistry 31, 3288-3293). Chemical depolarization of the transverse tubular system (T-tubule) moiety of labeled triads after appropriate priming induced first a rapid increase of the fluorescence intensity of the JFP-bound MCA probe, and then sarcoplasmic reticulum (SR) Ca2+ release. Upon increasing the magnitude of T-tubule depolarization by increasing the magnitude of T-tubule depolarization by increasing the degree of ionic replacement, both the amplitude of the MCA fluorescence change and the amount of released Ca2+ increased in parallel. Blockers of T-tubule-to-SR communication, such as nimodipine and low concentration of neomycin, inhibited both the MCA fluorescence change and the SR Ca2+ release. In contrast, the release blocking concentration of Mg2+ (2 mM) inhibited only SR Ca2+ release without affecting the fluorescence change. These results suggest that upon T-tubule depolarization the original state of the JFP (R) isomerizes to an activated state with higher MCA fluorescence (*R), which in turn changes into a subsequent state in which the release channel is open (*Ro):R-->*R-->*Ro.

Neomycin: a novel potent blocker of communication between T-tubule and sarcoplasmic reticulum.

Ca2+ release from the sarcoplasmic reticulum (SR) was induced in isolated triads by direct stimulation of the SR moiety by polylysine, or stimulation via chemical depolarization of the transverse tubule (T-tubule) moiety. Polylysine-induced release was blocked by neomycin with an IC50 (the concentration for half-maximal inhibition) of 0.3 microM. However, the IC50 for neomycin block of depolarization-induced Ca2+ release sharply decreased in a voltage-dependent fashion, and it was 5.3 nM at a maximal extent of T-tubule depolarization. These results suggest that the high affinity binding of neomycin to the triad leads to the specific blocking of the signal transmission from T-tubule to SR.

Chemical depolarization-induced SR calcium release in triads isolated from rabbit skeletal muscle.

Excitation-Ca2+ release coupling properties in the heavy microsomal fraction of the rabbit skeletal muscle enriched in triads were investigated by following the same type of approach used for the studies of excitation-contraction coupling in the skinned fiber system. Incubation of the triads with Mg-ATP in a solution containing 150 mM K+, 15.0-37.2 mM Na+, 150-180 mM gluconate-, and 150-200 microM Ca2+ (priming solution) led to (a) the generation of a T-tubule membrane potential making the cytoplasmic side negative, as assessed by potential-dependent uptake of the potential probe [14C]SCN- by triads, and (b) active transport of Ca2+ into the SR moiety. One volume of the primed (viz., polarized and Ca(2+)-loaded) triads was mixed with nine volumes of depolarization solution according to Cl(-)-replacement [Donaldson, S.K.B. (1985) J. Gen. Physiol 86, 501-525; Stephenson, E. W. (1985) J. Gen. Physiol. 86, 813-832] and Na(4)-replacement [Lamb, G.D., & Stephenson, D.G. (1990) J. Physiol. 423, 495-517] protocols used for the induction of contraction in skinned fiber system. The ionic replacement procedure by either protocol produced a rapid release of Ca2+ from SR as determined by stopped-flow fluorometry using fluo-3 as a Ca2+ probe in the presence of BAPTA-calcium buffer. Both the rate constant and the magnitude of Ca2+ release increased with the degree of ionic replacement. The ionic replacement-dependent changes in the release kinetics showed a striking similarity to the voltage-dependent changes of the Ca2+ transient in the intact fiber system.(ABSTRACT TRUNCATED AT 250 WORDS)

L-thyroxine activates the intracellular Ca2+ release channel of skeletal muscle sarcoplasmic reticulum.

L-thyroxine activated the Ca2+ release channel (ryanodine receptor) of skeletal muscle. [3H]ryanodine binding was stimulated by L-thyroxine in a dose dependent manner producing a two-fold increase at 250 microM. The same concentration induced a release of approximately 40% of the 45Ca2+ passively loaded into sarcoplasmic reticulum in 100 msec. Ca2+ release channel activity monitored in planar bilayers increased in the presence of 250 microM L-thyroxine from a control open probability of 0.02 +/- 0.03 to 0.17 +/- 0.12. Thyroid hormones may directly open Ca2+ release channels of skeletal muscle, thus altering intracellular Ca2+ homeostasis.