Evidence for a direct interaction between internal tetra-alkylammonium cations and the inactivation gate of cardiac sodium channels

Abstract

The effects of internal tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) were studied on human cardiac sodium channels (hH1) expressed in a mammalian tsA201 cell line. Outward currents were measured at positive voltages using a reversed Na gradient. TBA and TPeA cause a concentration- dependent increase in the apparent rate of macroscopic Na current inactivation in response to step depolarizations. At TPeA concentrations <50 μM the current decay is well fit by a single exponential over a wide voltage range. At higher concentrations a second exponential component is observed, with the fast component being dominant. The blocking and unblocking rate constants of TPeA were estimated from these data, using a three-state kinetic model, and were found to be voltage dependent. The apparent inhibition constant at 0 mV is 9.8 μM, and the blocking site is located 41 ± 3% of the way into the membrane field from the cytoplasmic side of the channel. Raising the external Na concentration from 10 to 100 mM reduces the TPeA-modified inactivation rates, consistent with a mechanism in which external Na ions displace TPeA from its binding site within the pore. TBA (500 μM) and TPeA (20 μM) induce a use-dependent block of Na channels characterized by a progressive, reversible, decrease in current amplitude in response to trains of depolarizing pulses delivered at 1-s intervals. Tetrapropylammonium (TPrA), a related symmetrical tetra-alkylammonium (TAA), blocks Na currents but does not alter inactivation (O'Leary, M. E., and R. Horn. 1994. Journal of General Physiology, 104:507-522.) or show use dependence. Internal TPrA antagonizes both the TPeA-induced increase in the apparent inactivation rate and the use dependence, suggesting that all TAA compounds share a common binding site in the pore. A channel blocked by TBA or TPeA inactivates at nearly the normal rate, but recovers slowly from inactivation, suggesting that TBA or TPeA in the blocking site can interact directly with a cytoplasmic inactivation gate.