CONTROL OF GAP JUNCTION CONDUCTANCE BY CALCINEURIN-DEPENDENT CX-43 PHOSPHORYLATION: IMPLICATIONS OF ARRHYTHMOGENESIS. (21st November 2014) - Waheed, A
Abstract : Slower action potential conduction, and consequent arrhythmias, follow decreased gap junction conductance, Gj. We investigated how the Ca2+-dependent phosphatase, calcineurin, regulates Gj through altering the phosphorylation of Cx43 protein, through a direct or indirect pathway. Gj was estimated in guinea-pig ventricular myocardium from the frequency-dependent intracellular impedance1, in physiological (Na=147.4 mM) and low-Na solutions (Na=29.4 mM; used to raise intracellular [Ca2+]). Calcineurin inhibitors (cyclosporine-A, 5 µM; CAIP, 50 µM) and an inhibitor of the Ca2+-independent phosphatase PP1 (tautomycin, 5 nM) were used. Western blots of tissue lysates measured: 1) total-Cx43; 2) Cx43 phosphoserineS365 and phosphoserineS368, normalised to total-Cx43; 3) total and phosphothreonine 35-I1inhibitor-1 (I1; a PP1 regulator), normalised to GAPDH. Means (±SEM) were compared by ANOVA, the null hypothesis rejected at p<0.05. Low-Na decreased Gj (59.8±3.6% control); reversed by CysA (85.5±5.5%; n=8) or CAIP (109.8±23.1%; n=3). Low-Na also decreased Cx43 phosphoserine365 vs control (0.57±0.02 vs 0.95±0.03; n=3), again reversed by cyclosporine-A (0.72±0.06, n=3; p<0.001) or CAIP (0.87±0.02, n=3). By contrast, Cx43 phosphoserine368 increased in low-Na (0.90±0.03 vs 0.08±0.02; n=6); also reversed by cyclosporine-A (0.15±0.9, n=6) and CAIP (0.13±0.03; n=6). Low-Na decreased phosphothreonine35-I1 (0.25±0.02 vs 0.83±0.00; n=6); reversed by CysA (0.37±0.02, n=6) and CAIP (0.71±0.08, n=6), suggesting calcineurin dephosphorylated phosphothreonine35-I1 and activated PP1. Tautomycin showed similar effects to calcineurin inhibitors in low-Na: reversing both the Gj decrease and alterations to phosphoserine365 and phosphoserine368. Raised [Ca2+]i decreases Gj by a calcineurin-dependent pathway via dephosphorylating phosphoserine365-Cx43, and subsequently phosphorylating serine368. Phosphoserine365-Cx43 is targeted by PP1 through calcineurin-dependent pathway.