http://nova.newcastle.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1802 In resting muscle, cytoplasmic Mg²⁺ is a potent inhibitor of Ca²⁺ release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca²⁺ sites (A-sites) thus preventing Ca²⁺ activation. We investigate the effects of luminal and cytoplasmic Ca²⁺ on Mg²⁺ inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg²⁺ inhibits RyRs at the A-site in the absence of Ca²⁺, indicating that Mg²⁺ is an antagonist and does not simply prevent Ca²⁺ activation. Cytoplasmic Ca²⁺ and Cs⁺ decreased Mg²⁺ affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca²⁺ regulation of Ca²⁺ release whereby increasing luminal [Ca²⁺] decreases the A-site affinity for cytoplasmic Mg²⁺ by a noncompetitive, allosteric mechanism that is independent of Ca²⁺ flow. Ryanodine increases the Ca²⁺ sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca²⁺, indicating that ryanodine activates independently of Ca²⁺. We describe a model for ion binding at the A-sites that predicts that modulation of Mg²⁺ inhibition by luminal Ca²⁺ is a significant regulator of Ca²⁺ release from the SR. We detected coupled gating of RyRs due to luminal Ca²⁺ permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca²⁺ and cytoplasmic Mg²⁺ did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca²⁺ was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca²⁺ emanating from their own pore but sensitive to Ca²⁺ from neighboring channels. 2010-04-27T06:11:00.204Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5571 The clustering of cardiac RyR mutations, linked to sudden cardiac death (SCD), into several regions in the amino acid sequence underlies the hypothesis that these mutations interfere with stabilising interactions between different domains of the RyR2. SCD mutations cause increased channel sensitivity to cytoplasmic and luminal Ca²⁺. A synthetic peptide corresponding to part of the central domain (DPc10:²⁴⁶⁰ G-P²⁴⁹⁵) was designed to destabilise the interaction of the N-terminal and central domains of wild-type RyR2 and mimic the effects of SCD mutations. With Ca²⁺ as the sole regulating ion, DPc10 caused increased channel activity which could be reversed by removal of the peptide whereas in the presence of ATP DPc10 caused no activation. In support of the domain destablising hypothesis, the corresponding peptide (DPc10-mut) containing the CPVT mutation R2474S did not affect channel activity under any circumstances. DPc10-induced activation was due to a small increase in RyR2 sensitivity to cytoplasmic Ca²⁺ and a large increase in the magnitude of luminal Ca²⁺ activation. The increase in the luminal Ca²⁺ response appeared reliant on the luminal-to-cytoplasmic Ca²⁺ flux in the channel, indicating that luminal Ca²⁺ was activating the RyR2 via its cytoplasmic Ca²⁺ sites. DPc10 had no significant effect on the RyR2 gating associated with luminal Ca2+ sensing sites. The results were fitted by the luminal-triggered Ca²⁺ feed-through model and the effects of DPc10 were explained entirely by perturbations in cytoplasmic Ca²⁺ -activation mechanism. 2010-04-27T04:40:01.020Z ]]>