Modification of cardiac Na⁺ channels by anthopleurin-A: effects on gating and kinetics

We used the whole cell patch clamp technique to investigate the characteristics of modification of cardiac Na⁺ channel gating by the sea anemone polypeptide toxin anthopleurin-A (AP-A). Guinea pig ventricular myocytes were isolated enzymatically using a retrograde perfusion apparatus. Holding potential was -140 mV and test potentials ranged from -100 to + 40 mV (pulse duration 100 or 1000 ms). AP-A (50-100 nM) markedly slowed the rate of decay of Na⁺ current (I_Na) and increased peak I_Na conductance (g_Na) by 38±5.5% (mean±SEM, P < 0.001, n = 12) with little change in slope factor (n = 12) or voltage midpoint of the g_Na/V relationship after correction for spontaneous shifts. The voltage dependence of steady-state I_Na availability (h_∞) demonstrated an increase in slope factor from 5.9±0.8 mV in control to 8.0±0.7 mV after modification by AP-A (P < 0.01, n = 14) whereas any shift in the voltage midpoint of this relationship could be accounted for by a spontaneous time-dependent shift. AP-A-modified I_Na showed a use-dependent decrease in peak current amplitude (interpulse interval 500 ms) when pulse duration was 100 ms (-15±2%, P < 0.01, n = 17) but showed no decline when pulse duration was 100 ms (-3±1%). This use-dependent effect was probably the result of a decrease in the rate of recovery from inactivation caused by AP-A which had a small effect on the fast time constant of recovery (from 4.1±0.3 ms in control to 6.0±1.1 ms after AP-A, P < 0.05) but increased the slow time constant from 66.2±6.5 ms in control to 188.9±36.4 ms (P< 0.002, n = 19) after exposure to AP-A. Increasing external divalent cation concentration (either Ca²⁺ or Mg²⁺) to 10 mM abolished the effects of AP-A on the rate of I_Na decay. These results demonstrate that modification of cardiac Na⁺ channels by AP-A markedly slowed I_Na inactivation and altered the voltage dependence of activation; these alterations in gating characteristics, in turn, caused an increase in g_Na presumably by increasing the number of channels open at peak I_Na. AP-A slows the rate of recovery of I_Na from inactivation which is probably the basis for a use-dependent decrease in peak amplitude. Finally, AP-A binding is sensitive to external divalent cation concentrations. Thus, increasing [Mg²⁺]_o or [Ca²⁺]_o displaces AP-A from binding, suggesting that they share related binding sites on the external surface of the Na⁺ channel.