Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop.
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.
Muscle Research Group, John Curtin School of Medical Research, P.O. Box 334, Canberra, ACT 2601, Australia.
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Gallant
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Curtis
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Pace
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Dulhunty
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D007473:Ion Channels / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013997:Time Factors
D020746:Calcium Channels, L-Type; D007473:Ion Channels; D008051:Lipid Bilayers; D010455:Peptides; D019837:Ryanodine Receptor Calcium Release Channel; D001120:Arginine; D003545:Cysteine; D002118:Calcium