Characterization of 4-aminopyridine block of the transient outward K+ current in adult rat ventricular myocytes.

The blocking action of 4-aminopyridine (4-AP) on the transient outward K+ current (ITO) in isolated rat ventricular myocytes was studied using the whole-cell configuration of the patch-clamp technique. 4-AP inhibition of ITO was concentration-dependent with half-maximal block occurring at 0.2 mM. At high concentrations (> 1 mM), 4-AP appeared to slow both the activation and inactivation phases of ITO. This resulted in a crossover phenomenon where in the presence of 4-AP the outward current was less than control at the beginning of a depolarizing pulse but crossed over during the pulse to become greater than control. Inhibition of ITO by 4-AP was voltage-dependent. Steady-state block of ITO by 4-AP was greatest at or near resting membrane potentials (i.e., -70 mV) but decreased with membrane depolarization. The voltage-dependence of block was steep and was well described by a Boltzmann relationship with a slope factor of approximately 4 mV. The midpoint potential for block was dependent on the concentration of 4-AP, being -41.6 +/- 0.4 mV (n = 9), -40.7 +/- 1.3 mV (n = 6), -34.0 +/- 1.6 mV (n = 5) and -30.1 +/- 0.2 (n = 15) at 0.3, 1, 3 and 10 mM, respectively. The midpoint potential for activation was -12.6 mV and was -46.9 mV for inactivation. The concentration-dependence of the voltage-dependence of 4-AP block can be explained by assuming that the sequential closed states through which the channel passes during activation exhibit successively lower affinities for 4-AP. Onset of ITO block by 4-AP was slow. The association (kON) and dissociation. (kOFF) rate constants for binding at -70 mV were: kON = 207 M-1 s-1 and kOFF = 0.090 s-1. The time constant for unblock (tau UNBLOCK) of ITO at 0 mV was independent of 4-AP concentration indicating that there was no binding of 4-AP at this potential. kOFF (1/tau UNBLOCK) at 0 mV was 2.4 s-1 which is approximately 25-fold faster then at -70 mV. The results suggest that 4-AP binds most strongly to closed channels with the inactivation gate open. The conformational changes that occur during channel opening induce a decrease in affinity for 4-AP so that when the channel is in the open state, 4-AP binding is at its weakest. The processes of 4-AP block and inactivation appear to be mutually exclusive.