Abstract

It has been suggested that protein tyrosine kinase (PTK) activity can directly regulate cardiac L-type Ca²⁺ channels. This conclusion is based to a large extent on the observation that the PTK inhibitor genistein can inhibit the cardiac L-type Ca²⁺current. The purpose of the present study was to determine whether the ability of genistein to inhibit cardiac L-type Ca²⁺ channel activity is due to inhibition of PTK activity. Genistein significantly reduced the magnitude of the L-type Ca²⁺ current in guinea pig ventricular myocytes recorded using the whole-cell patch-clamp technique. However, this effect was associated with extracellular, not intracellular, application of the drug. Peroxovanadate (PVN), a potent protein tyrosine phosphatase inhibitor, had no effect on the basal Ca²⁺ current. PVN was also ineffective in preventing the inhibitory effect of genistein. Internal perfusion of cells with a pipette solution containing ATPγS was used to prevent reversibility of phosphorylation-dependent processes. This treatment did not alter the inhibitory effect of genistein, although it did result in irreversible protein kinase A-dependent regulation of the Ca²⁺ current. Bath application of lavendustin A, a PTK inhibitor that is structurally unrelated to genistein, did not affect the Ca²⁺ current amplitude. The inhibitory effect of genistein was also associated with a hyperpolarizing shift in the voltage dependence of Ca²⁺ channel inactivation. These results are consistent with the conclusion that the cardiac L-type Ca²⁺ current is not directly regulated by PTK activity and that the inhibitory effect of genistein is due to direct non-catalytic blockade of the channels.