RyR1-mediated Ca2+ Leak and Ca2+ Entry Determine Resting Intracellular Ca2+ in Skeletal Myotubes

José M Eltit,Tianzhong Yang,Hongli Li,Tadeusz F Molinski,Isaac N Pessah,Paul D Allen,José R Lopez

doi:10.1074/jbc.m110.107300

Abstract

The control of resting free Ca(2+) in skeletal muscle is thought to be a balance of channels, pumps, and exchangers in both the sarcolemma and sarcoplasmic reticulum. We explored these mechanisms using pharmacologic and molecular perturbations of genetically engineered (dyspedic) muscle cells that constitutively lack expression of the skeletal muscle sarcoplasmic reticulum Ca(2+) release channels, RyR1 and RyR3. We demonstrate here that expression of RyR1 is responsible for more than half of total resting Ca(2+) concentration ([Ca(2+)](rest)) measured in wild type cells. The elevated [Ca(2+)](rest) in RyR1-expressing cells is not a result of active gating of the RyR1 channel but instead is accounted for by the RyR1 ryanodine-insensitive Ca(2+) leak conformation. In addition, we demonstrate that basal sarcolemmal Ca(2+) influx is also governed by RyR1 expression and contributes in the regulation of [Ca(2+)](rest) in skeletal myotubes.

Highlights

In addition to the “classical” release pathway mediated by RyR1 activation, at rest there is ample evidence for the existence of a second less defined sarcoplasmic reticulum (SR) Ca2ϩ efflux pathway that has been referred to as ryanodine (Ry)-insensitive “Ca2ϩ leak” [4, 9, 10]
The purpose of this study was to examine whether the expression of RyR1 has any effect on [Ca2ϩ]rest, the resting Ca2ϩ entry, and SR Ca2ϩ loading in skeletal muscle
Our study demonstrates that expression of WtRyR1 is associated with a significant increase in [Ca2ϩ]rest, in resting Ca2ϩ entry, with no significant change in SR Ca2ϩ loading compared with NullRyR myotubes

Summary

Introduction

In addition to the “classical” release pathway mediated by RyR1 activation, at rest there is ample evidence for the existence of a second less defined SR Ca2ϩ efflux pathway that has been referred to as ryanodine (Ry)-insensitive “Ca2ϩ leak” [4, 9, 10]. This Ca2ϩ leak can be broadly defined as a passive efflux of Ca2ϩ from the SR under resting or quiescent conditions. Similar results were obtained in primary myotubes generated from RyR1/3-null dyspedic mice and their wild type littermates

Objectives

Methods

Results

Conclusion