Irreversible block of cardiac mutant Na+ channels by batrachotoxin

Abstract

Batrachotoxin (BTX) not only keeps the voltage-gated Na+ channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na+ channels, how Na+ ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na+ ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNav1.5 Na+ channels. We found that BTX at 5 mM indeed elicited a strong block of hNav1.5-N927K currents (~70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na+ channel gating. Once occurred, this unique use-dependent block of hNav1.5-N927K Na+ channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNav1.5-N927K Na+ channels albeit with a faster on-rate; ~90% of peak Na+ currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNav1.5-N927K Na+ channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/ H/D/S/Q/G/E substitutions in fast inactivation-deficient hNav1.5 Na+ channels, only N927K/R Na+ currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNav1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na+ ions.