Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2 > A1 > A1S > NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by approximately 30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at -40 mV, with 10 microM A1 or 50 nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100 nM cis Ca2+. Inhibition by A1 was greatest at +40 mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100 microM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.

SummaryAlternative splicing of ASI residues (Ala 3481 -Gln 3485 ) in the skeletal muscle ryanodine receptor (RyR1) is developmentally regulated: the residues are present in adult ASI(+)RyR1, but absent in the juvenile ASI(-)RyR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RyR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr 3471 -Gly 3500 ) in the splice variants. Depolarisation-dependent Ca 2+ release was enhanced by >50% in myotubes expressing ASI(-)RyR1 compared with ASI(+)RyR1, although DHPR L-type currents and SR Ca 2+ content were unaltered, while ASI(-)RyR1 channel function was actually depressed. The effect on EC coupling did not depend on changes in ASI domain secondary structure. Probing RyR1 function with peptides possessing the ASI domain sequence indicated that the domain contributes to an inhibitory module in RyR1. The action of the peptide depended on a sequence of basic residues and their alignment in an α-helix adjacent to the ASI splice site. This is the first evidence that the ASI residues contribute to an inhibitory module in RyR1 that influences EC coupling. Implications for development and DM1 are discussed.

Phosphorylation of skeletal muscle ryanodine receptor (RyR) calcium release channels by endogenous kinases incorporated into lipid bilayers with native sarcoplasmic reticulum vesicles was investigated during exposure to 2 mM cytoplasmic ATP. Activation of RyRs after 1-min exposure to ATP was reversible upon ATP washout. In contrast, activation after 5 to 8 min was largely irreversible: the small fall in activity with washout was significantly less than that after brief ATP exposure. The irreversible activation was reduced by acid phosphatase and was not seen after exposure to nonhydrolyzable ATP analogs. The data suggested that the channel complex was phosphorylated after addition of ATP and that phosphorylation reduced the RyR's sensitivity to ATP, adenosine, and Ca(2+). The endogenous kinase was likely to be a calcium calmodulin kinase II (CaMKII) because the CaMKII inhibitor KN-93 and an inhibitory peptide for CaMKII prevented the phosphorylation-induced irreversible activation. In contrast, phosphorylation effects remained unchanged with inhibitory peptides for protein kinase C and A. The presence of CaMKIIbeta in the SR vesicles was confirmed by immunoblotting. The results suggest that CaMKII is anchored to skeletal muscle RyRs and that phosphorylation by this kinase alters the enhancement of channel activity by ATP and Ca(2+).

Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca2+ store where it modifies Ca2+ signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca2+ release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca2+ signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1–S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.

The aim of the present study was to examine residues that are variably spliced in the juvenile and adult isoforms of the skeletal-muscle RyR1 (type 1 ryanodine receptor). The juvenile ASI(-) splice variant is less active than the adult ASI(+) variant and is overexpressed in patients with DM (myotonic dystrophy) [Kimura, Nakamori, Lueck, Pouliquin, Aoike, Fujimura, Dirksen, Takahashi, Dulhunty and Sakoda (2005) Hum. Mol. Genet. 14, 2189-2200]. In the present study, we explore the ASI region using synthetic peptides corresponding to rabbit RyR1 residues Thr3471-Gly3500 either containing [PASI(+)] or lacking [PASI(-)] the ASI residues. Both peptides increased [3H]ryanodine binding to rabbit RyR1s, increased Ca2+ release from sarcoplasmic reti-culum vesicles and increased single RyR1 channel activity. The peptide PASI(-) was more active in each case than PASI(+). [3H]Ryanodine binding to recombinant ASI(+)RyR1 or ASI(-)-RyR1 was enhanced more by PASI(-) than PASI(+), with the greatest increase seen when PASI(-) was added to ASI(-)RyR1. The activation of the RyR channels is consistent with the hypo-thesis that the peptides interrupt an inhibitory inter-domain inter-action and that PASI(-) is more effective at interrupting this interaction than PASI(+). We therefore suggest that the ASI(-) sequence interacts more tightly than the ASI(+) sequence with its binding partner, so that the ASI(-)RyR1 is more strongly inhibited (less active) than the ASI(+)RyR1. Thus the affinity of the binding partners in this inter-domain interaction may deter-mine the activities of the mature and juvenile isoforms of RyR1 and the stronger inhibition in the juvenile isoform may contribute to the myopathy in DM.

The ryanodine receptor (RyR) is a large conductance ion channel which allows Ca¥ release from the sarcoplasmic reticulum (SR) of skeletal muscle to initiate contraction in response to sarcolemmal depolarization (Dulhunty, 1992).The contraction is terminated when Ca¥ is pumped back into the SR by the Ca¥-Mg¥-ATPase. The functional skeletal RyR Ca¥-release channel is a homotetrameric complex of RyR monomers (Mr •560000) and four FK506-Journal of Physiology (1999), 514.2, pp. 313-326 313 Effects of ivermectin and midecamycin on ryanodine receptors and the Ca¥-ATPase in sarcoplasmic reticulum of rabbit and rat skeletal muscle 0·66-40 ìÒ) activated six of eight native, four of four control-incubated and eleven of eleven FKBP12-'stripped' RyR channels. Midecamycin (cis, 10-30 ìÒ) activated three of four single native channels, six of eight control-incubated channels and six of seven FKBP12-stripped channels. Activity declined when either drug was washed out. 3. Neither ivermectin nor midecamycin removed FKBP12 from RyRs. Western blots of terminal cisternae (TC), incubated for 15 min at 37°C with 40 ìÒ ivermectin or midecamycin, showed normal amounts of FKBP12. In contrast, no FKBP12 was detected after incubation with 40 ìÒ rapamycin. 4. Ivermectin reduced Ca¥ uptake by the SR Ca¥-Mg¥-ATPase. Ca¥ uptake by TC fell to•40% in the presence of ivermectin (10 ìÒ), both with and without 10 ìÒ Ruthenium Red. Ca¥ uptake by longitudinal SR also fell to •40% with 10 ìÒ ivermectin. Midecamycin (10 ìÒ) reduced Ca¥ uptake by TC vesicles to •76% without Ruthenium Red and to •90% with Ruthenium Red. 5. The rate of rise of extravesicular [Ca¥] increased •2-fold when 10 ìÒ ivermectin was added to TC vesicles that had been partially loaded with Ca¥ and then Ca¥ uptake blocked by 200 nÒ thapsigargin. Ivermectin also potentiated caffeine-induced Ca¥ release to •140% of control. These increases in Ca¥ release were not seen with midecamycin. 6. Ivermectin, but not midecamycin, reversibly reduced Ca¥ loading in four of six skinned rat extensor digitorum longus (EDL) fibres to •90%, and reversibly increased submaximal caffeine-induced contraction in five of eight fibres by •110% of control. Neither ivermectin nor midecamycin altered twitch or tetanic tension in intact EDL muscle fibres within 20 min of drug addition. 7. The results confirm the hypothesis that compounds with a macrocyclic lactone ring structure can directly activate RyRs. Unexpectedly, ivermectin also reduced Ca¥ uptake into the SR. These effects of ivermectin on SR Ca¥ handling may explain some effects of the macrolide drugs on mammals.

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.

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