https://nova.newcastle.edu.au/vital/access/ /manager/Index en-au 5 https://nova.newcastle.edu.au/vital/access/ /manager/Repository/uon:34969 d = 121 ± 14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca²⁺-spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.]]> Wed 24 Jun 2020 11:42:42 AEST ]]> https://nova.newcastle.edu.au/vital/access/ /manager/Repository/uon:15961 Wed 11 Apr 2018 14:53:16 AEST ]]> https://nova.newcastle.edu.au/vital/access/ /manager/Repository/uon:53989 Thu 25 Jan 2024 13:03:51 AEDT ]]> https://nova.newcastle.edu.au/vital/access/ /manager/Repository/uon:19035 Sat 24 Mar 2018 08:05:26 AEDT ]]> https://nova.newcastle.edu.au/vital/access/ /manager/Repository/uon:18980 i = 10 µM at 100 nM Ca²⁺) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca²⁺, RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg²⁺ inhibition than those from sheep and rat. The Kₐ values for luminal Ca²⁺ activation were similar in the three species (35 µM for human, 12 µM for sheep, and 10 µM for rat). From the relationship between open probability and luminal [Ca²⁺], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ~10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg²⁺ as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca²⁺ and Mg²⁺ only occurred when cytoplasmic [Ca²⁺] was <3 µM. The activation response of RyR2 to luminal and cytoplasmic Ca²⁺ was strongly dependent on the Mg²⁺ concentration. Addition of physiological levels (1 mM) of Mg²⁺ raised the Kₐ for cytoplasmic Ca²⁺ to 30 µM (human and sheep) or 90 µM (rat) and raised the Kₐ for luminal Ca²⁺ to ~1 mM in all species. This is the first report of the regulation by Ca²⁺ and Mg²⁺ of native RyR2 receptor activity from healthy human hearts.]]> Sat 24 Mar 2018 07:58:52 AEDT ]]> https://nova.newcastle.edu.au/vital/access/ /manager/Repository/uon:22802 2+ release. Although dantrolene inhibits Ca²⁺ release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca²⁺ release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca²⁺] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of P_o (E_max) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC₅₀ of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC₅₀ = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca²⁺ release in the absence of CaM, but reduced Ca²⁺ wave frequency (IC₅₀ = 0.42 ± 0.18 µM, E_max = 47 ± 4%) and amplitude (IC₅₀ = 0.19 ± 0.04 µM, E_max = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.]]> Sat 24 Mar 2018 07:12:18 AEDT ]]> https://nova.newcastle.edu.au/vital/access/ /manager/Repository/uon:33275 Mon 24 Sep 2018 13:26:23 AEST ]]>