Regulation of ryanodine receptors by calsequestrin: effect of high luminal Ca²⁺ and phosphorylation

¹ Australian National University
² Rurh Universitat
³ University of Newcastle
⁴ Australia National University

^* To whom correspondence should be addressed. E-mail: nicole.beard{at}anu.edu.au.

Submitted on August 24, 2004
Revised on October 26, 2004
Accepted on 15 February 2005

	Abstract

Calsequestrin, the major calcium sequestering protein in the sarcoplasmic reticulum of muscle, forms a quaternary complex with the ryanodine receptor calcium release channel, and the intrinsic membrane proteins triadin and junctin. We have investigated the possibility that calsequestrin is a luminal calcium concentration sensor for the ryanodine receptor. We measured the luminal calcium concentration at which calsequestrin dissociates from the ryanodine receptor and the effect of calsequestrin on the response of the ryanodine receptor to changes in luminal calcium. We provide electrophysiological and biochemical evidence that 1) luminal calcium concentration of 4 mM dissociates calsequestrin from junctional face membrane, while in the range of 1 to 3 mM calsequestrin remains attached, 2) the association with calsequestrin inhibits ryanodine receptor activity, but amplifies its response to changes in luminal calcium concentration, and 3) under physiological calcium conditions (1 mM), phosphorylation of calsequestrin does not alter its ability to inhibit native ryanodine receptor activity when the anchoring proteins triadin and junctin are present. These data suggest that the quaternary complex is intact in vivo, and provides further evidence that calsequestrin is involved in the sarcoplasmic reticulum calcium signaling pathway, and has a role as a luminal calcium sensor for the ryanodine receptor.

Key Words: calcium binding proteins, junctin, sarcoplasmic reticulum, skeletal muscle, triadin

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