REGULATION OF SKELETAL-MUSCLE CA2+ RELEASE CHANNEL (RYANODINE RECEPTOR) BY CA2+ AND MONOVALENT CATIONS AND ANIONS

The effects of ionic composition and strength on rabbit skeletal muscl e Ca2+ release channel (ryanodine receptor) activity were investigated in vesicle-Ca-45(2+) flux, single channel and [H-3]ryanodine binding measurements. In <0.01 mu M Ca2+ media, the highest Ca-45(2+) efflux r ate was measured in 0.25 M choline-Cl medium followed by 0.25 M KCl, c holine 4-morpholineethanesulfonic acid (Mes), potassium 1,4-piperazine diethanesulfonic acid (Pipes), and K-Mes medium. In all five media, th e Ca-45(2+) efflux rates were increased when the free [Ca2+] was raise d from <0.01 mu M to 20 mu M and decreased as the free [Ca2+] was furt her increased to 1 mM. An increase in [KCl] augmented Ca2+-gated singl e channel activity and [H-3]ryanodine binding. In [H-3]ryanodine bindi ng measurements, bell-shaped Ca2+ activation/inactivation curves were obtained in media containing different monovalent cations (Li+, Na+ K, Cs+, and choline(+)) and anions (Cl-, Mes(-), and Pipes(-)). In chol ine-Cl medium, substantial levels of [H-3]ryanodine binding were obser ved at [Ca2+] <0.01 mu M. Replacement of Cl- by Mes(-) or Pipes(-) red uced [H-3]ryanodine binding levels at all [Ca2+]. In all media, the Ca 2+-dependence of [H-3]ryanodine binding could be well described assumi ng that the skeletal muscle ryanodine receptor possesses cooperatively interacting high-affinity Ca2+ activation and low-affinity Ca2+ inact ivation sites. AMP primarily affected [H-3]ryanodine binding by decrea sing the apparent affinity of the Ca2+ inactivation site(s) for Ca2+, while caffeine increased the apparent affinity of the Ca2+ activation site for Ca2+. Competition studies indicated that ionic composition af fected Ca2+-dependent receptor activity by at least three different me chanisms: (i) competitive binding of Mg2+ and monovalent cations to th e Ca2+ activation sites, (ii) binding of divalent cations to the Ca2inactivation sites, and (iii) binding of anions to specific anion regu latory sites.