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Myoplasmic resting Ca2+ regulation by ryanodine receptors is under the control of a novel Ca2+-binding region of the receptor.

ABSTRACT: Passive SR (sarcoplasmic reticulum) Ca2+ leak through the RyR (ryanodine receptor) plays a critical role in the mechanisms that regulate [Ca2+]rest (intracellular resting myoplasmic free Ca2+ concentration) in muscle. This process appears to be isoform-specific as expression of either RyR1 or RyR3 confers on myotubes different [Ca2+]rest. Using chimaeric RyR3-RyR1 receptors expressed in dyspedic myotubes, we show that isoform-dependent regulation of [Ca2+]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 (Ca2+ leak regulatory) region. [Ca2+]rest regulation by the CLR region was associated with alteration of RyRs' Ca2+-activation profile and changes in SR Ca2+-leak rates. Biochemical analysis using Tb3+-binding assays and intrinsic tryptophan fluorescence spectroscopy of purified CLR domains revealed that this determinant of RyRs holds a novel Ca2+-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 Ca2+ leak and confer on RyR1 and RyR3 distinctive [Ca2+]rest regulatory properties. The identification of a new Ca2+-binding domain of RyRs with a key modulatory role in [Ca2+]rest regulation provides new insights into Ca2+-mediated regulation of RyRs.

SUBMITTER: Chen Y

PROVIDER: S-EPMC4019983 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Ca2+ leak through ryanodine receptor 1 regulates thermogenesis in resting skeletal muscle.

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Project description:Ryanodine receptors (RyRs) are Ca2+ release channels in the sarcoplasmic reticulum of skeletal and cardiac muscles and are essential for muscle contraction. Mutations in genes encoding RyRs cause various muscle and arrhythmogenic heart diseases. Although RyR channels are activated by Ca2+, the actual mechanism of Ca2+ binding remains largely unknown. Here, we report the molecular basis of Ca2+ binding to RyRs for channel activation and discuss its implications in disease states. RyR1 and RyR2 carrying mutations in putative Ca2+ and caffeine-binding sites were functionally analysed. The results were interpreted with respect to recent near-atomic resolution RyR1 structures in various ligand states. We demonstrate that a tryptophan residue in the caffeine-binding site controls the structure of the Ca2+-binding site to regulate the Ca2+ sensitivity. Our results reveal the initial step of RyR channel activation by Ca2+ and explain the molecular mechanism of Ca2+ sensitization by caffeine and disease-causing mutations.

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Project description:Spontaneous Ca(2+) release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca(2+) release (SOICR) can result in Ca(2+) waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here we show that a point mutation, E4872A, in the helix bundle crossing region (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, Ca(2+) activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni(2+)-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca(2+)-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca(2+), explaining the regulation of RyR2 by luminal Ca(2+), the initiation of Ca(2+) waves and Ca(2+)-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.

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Project description:In cardiac muscle, Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) is mediated by ryanodine receptor (RyR) Ca(2+) release channels. The inherent positive feedback of CICR is normally well-controlled. Understanding this control mechanism is a priority because its malfunction has life-threatening consequences.We show that CICR local control is governed by SR Ca(2+) load, largely because load determines the single RyR current amplitude that drives inter-RyR CICR.We differentially manipulated single RyR Ca(2+) flux amplitude and SR Ca(2+) load in permeabilized ventricular myocytes as an endogenous cell biology model of the heart. Large RyR-permeable organic cations were used to interfere with Ca(2+) conductance through the open RyR pore. Single-channel studies show this attenuates current amplitude without altering other aspects of RyR function. In cells, the same experimental maneuver increased resting SR Ca(2+) load. Despite the increased load, Ca(2+) spark (inter-RyR CICR events) frequency decreased and sparks terminated earlier.Spark local control follows single RyR current amplitude, not simply SR Ca(2+) load. Spark frequency increases with load because spontaneous RyR openings at high loads produce larger currents (ie, a larger CICR trigger signal). Sparks terminate when load falls to the point at which single RyR current amplitude is no longer sufficient to sustain inter-RyR CICR. Thus, RyRs that spontaneously close no longer reopen and local Ca(2+) release ends.

| S-EPMC3417769 | biostudies-literature

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