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 muscle 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 rate was measured in 0.25 M choline-Cl medium followed by 0.25 M KCl, choline 4-morpholineethanesulfonic acid (Mes), potassium 1,4-piperazinediethanesulfonic acid (Pipes), and K-Mes medium. In all five media, the Ca-45(2+) efflux rates were increased when the free [Ca2+] was raised from <0.01 mu M to 20 mu M and decreased as the free [Ca2+] was further increased to 1 mM. An increase in [KCl] augmented Ca2+-gated single channel activity and [H-3]ryanodine binding. In [H-3]ryanodine binding 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 choline-Cl medium, substantial levels of [H-3]ryanodine binding were observed at [Ca2+] <0.01 mu M. Replacement of Cl- by Mes(-) or Pipes(-) reduced [H-3]ryanodine binding levels at all [Ca2+]. In all media, the Ca2+-dependence of [H-3]ryanodine binding could be well described assuming that the skeletal muscle ryanodine receptor possesses cooperatively interacting high-affinity Ca2+ activation and low-affinity Ca2+ inactivation sites. AMP primarily affected [H-3]ryanodine binding by decreasing 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 affected Ca2+-dependent receptor activity by at least three different mechanisms: (i) competitive binding of Mg2+ and monovalent cations to the Ca2+ activation sites, (ii) binding of divalent cations to the Ca2+ inactivation sites, and (iii) binding of anions to specific anion regulatory sites.