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 (ICa) 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 ICa at the t-tubules. Ventricular myocytes were isolated from male Wistar rats and detubulated using formamide. ICa 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 μmol/L) and the CaMKII inhibitor KN-93 (5 μmol/L) decreased the amplitude of ICa in intact cells but had no effect on ICa 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 ICa 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.