Localised Ca channel phosphorylation modulates the distribution of L-type Ca current in cardiac myocytes

The t-tubule network is central to excitation-contraction coupling in mammalian cardiac ventricular myocytes, with recent studies showing that the majority of Ca influx via the L-type Ca current (I-Ca) occurs across the t-tubule membrane The present study investigated whether tonic phosphorylation of the L-type Ca channel is different at the t-tubule and surface membranes, and if this could account for the high density of I-Ca at the t-tubules Ventricular myocytes were isolated from male Wistar rats and detubulated using formamide. I-Ca was recorded using the whole cell patch clamp technique, and Ca transients were recorded using fluo-3 in conjunction with confocal microscopy The protein kinase A (PKA) inhibitor H-89 (10 mu mol/L) and the CaMKII inhibitor KN-93 (5 mu mol/L) decreased the amplitude of I-Ca in intact cells but had no effect on I-Ca amplitude in detubulated cells These inhibitors also decreased the amplitude of the Ca transient in intact cells but not in detubulated cells Antibody staining for phosphorylated L-type Ca channel showed significantly higher phosphorylation at the t-tubules than at the surface membrane in intact cells. Thus it appears that tonic phosphorylation of the L-type Ca channel maintains the amplitude of I-Ca and occurs predominantly at the t-tubules This may have important implications in heart disease, in which changes of phosphorylation and t-tubule density have been reported (C) 2010 Elsevier Ltd All rights reserved