Myoplasmic resting Ca(2+) regulation by ryanodine receptors is under the control of a novel Ca(2+)-binding region of the receptor

Passive SR (sarcoplasmic reticulum) Ca(2+) leak through the RyR (ryanodine receptor) plays a critical role in the mechanisms that regulate [Ca(2+)](rest) (intracellular resting myoplasmic free Ca(2+) concentration) in muscle. This process appears to be isoform-specific as expression of either RyR1 or RyR3 confers on myotubes different [Ca(2+)](rest). Using chimaeric RyR3–RyR1 receptors expressed in dyspedic myotubes, we show that isoform-dependent regulation of [Ca(2+)](rest) is primarily defined by a small region of the receptor encompassing amino acids 3770–4007 of RyR1 (amino acids 3620–3859 of RyR3) named as the CLR (Ca(2+) leak regulatory) region. [Ca(2+)](rest) regulation by the CLR region was associated with alteration of RyRs’ Ca(2+)-activation profile and changes in SR Ca(2+)-leak rates. Biochemical analysis using Tb(3+)-binding assays and intrinsic tryptophan fluorescence spectroscopy of purified CLR domains revealed that this determinant of RyRs holds a novel Ca(2+)-binding domain with conformational properties that are distinctive to each isoform. Our data suggest that the CLR region provides channels with unique functional properties that modulate the rate of passive SR Ca(2+) leak and confer on RyR1 and RyR3 distinctive [Ca(2+)](rest) regulatory properties. The identification of a new Ca(2+)-binding domain of RyRs with a key modulatory role in [Ca(2+)](rest) regulation provides new insights into Ca(2+)-mediated regulation of RyRs.