Altered binding site for Ca2+ in the ryanodine receptor of human malignant hyperthermia.
The binding properties of [3H]ryanodine, a specific ligand of the receptor complex that forms the Ca2+ release channel of sarcoplasmic reticulum, were studied in normal (N) and malignant hyperthermia-susceptible (MH) human skeletal muscle. Integrity of the solubilized ryanodine receptor was demonstrated by single-channel recordings in planar bilayers and by the changes produced by activators and inhibitors of the Ca2+ release channel on the binding properties of [3H]ryanodine. N and MH receptors were capable of binding [3H]ryanodine in a Ca(2+)-dependent manner. Scatchard analysis showed that a single binding site for [3H]ryanodine was present in either N or MH muscle. Binding affinity was approximately the same in N and MH (Kd approximately 7 nM), when the Ca2+ concentration was greater than 30 microM. At 0.3 microM Ca2+, MH receptors displayed a higher affinity for [3H]ryanodine (Kd = 4.1 +/- 1.0 nM) than N receptors (Kd = 7.1 +/- 0.8 nM). The presence of a single Kd for [3H]ryanodine in MH muscle, distinct from that of N muscle, indicated that MH muscle does not have detectable levels of N receptors. Ca2+ dependence of [3H]ryanodine binding further suggested that MH receptors had a higher affinity for Ca2+ (Kd[Ca2+] = 120 +/- 50 nM) than N receptors (Kd[Ca2+] = 250 +/- 80 nM). Caffeine increased [3H]ryanodine binding at submicromolar [Ca2+], and the effect was larger in MH. Apparent affinity constants for caffeine were 13 +/- 1.8 mM in N and 6 +/- 0.8 mM in MH receptors. Evidently, the ryanodine receptor of MH-susceptible human skeletal muscle has an unusually high sensitivity to Ca2+ which is augmented by caffeine.(ABSTRACT TRUNCATED AT 250 WORDS)
Valdivia
H H
HH
Department of Physiology, University of Wisconsin Medical School, Madison 53706.
Hogan
K
K
Coronado
R
R
D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D004056:Differential Threshold; D006801:Humans; D008305:Malignant Hyperthermia / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism* / Q000502:physiology; D012016:Reference Values; D019837:Ryanodine Receptor Calcium Release Channel
D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002118:Calcium