Regulation of Skeletal Muscle Ca2+ Release Channel (Ryanodine Receptor) by Ca2+ and Monovalent Cations and Anions

Gerhard Meissner,Eduardo Rios,Ashutosh Tripathy,Daniel A Pasek

doi:10.1074/jbc.272.3.1628

Abstract

The effects of ionic composition and strength on rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) activity were investigated in vesicle-45Ca2+ flux, single channel and [3H]ryanodine binding measurements. In <0.01 microM Ca2+ media, the highest 45Ca2+ 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 45Ca2+ efflux rates were increased when the free [Ca2+] was raised from <0.01 microM to 20 microM and decreased as the free [Ca2+] was further increased to 1 mM. An increase in [KCl] augmented Ca2+-gated single channel activity and [3H]ryanodine binding. In [3H]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 [3H]ryanodine binding were observed at [Ca2+] <0.01 microM. Replacement of Cl- by Mes- or Pipes- reduced [3H]ryanodine binding levels at all [Ca2+]. In all media, the Ca2+-dependence of [3H]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 [3H]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.

Highlights

In skeletal muscle, an intracellular Ca2ϩ conducting channel releases Ca2ϩ from the sarcoplasmic reticulum (SR)1 in response to an action potential, to bring about muscle contraction [1,2,3]
SR Vesicle-45Ca2ϩ Efflux Measurements—In preliminary experiments, the effects of ionic composition on Ca2ϩ release channel activity were assessed in SR vesicle-45Ca2ϩ efflux measurements
Vesicles were passively loaded with 2 mM 45Ca2ϩ in 0.25 M KCl or 0.25 M choline-Cl medium and diluted into the same medium containing 5 mM Mg2ϩ and Ͻ0.01 ␮M free Ca2ϩ, Ͻ0.01 ␮M free Ca2ϩ, or 20 ␮M free Ca2ϩ. 45Ca2ϩ efflux was slow when vesicles were diluted into media containing Ͻ0.01 ␮M Ca2ϩ and 5 mM Mg2ϩ

Summary

Introduction

An intracellular Ca2ϩ conducting channel releases Ca2ϩ from the sarcoplasmic reticulum (SR) in response to an action potential, to bring about muscle contraction [1,2,3]. Skeletal muscle RyR activity is affected by the ionic strength and composition of the assay media. Anions often classified as chaotropic ions (Cl04Ϫ, SCNϪ, IϪ, NO3Ϫ) [13, 14] and inorganic phosphate anions [15] stimulate Ca2ϩ release channel activity and [3H]ryanodine binding, whereas replacement of ClϪ by gluconateϪ decreases SR Ca2ϩ release and [3H]ryanodine binding [13]. These results suggest that monovalent cations and anions as well as osmolarity or viscosity may modulate skeletal muscle. The mechanism(s) by which these ions affect the SR Ca2ϩ release channel have remained unclear

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