THE CALCIUM-INDEPENDENT TRANSIENT OUTWARD POTASSIUM CURRENT IN ISOLATED FERRET RIGHT VENTRICULAR MYOCYTES .2. CLOSED STATE REVERSE USE-DEPENDENT BLOCK BY 4-AMINOPYRIDINE

Block of the calcium-independent transient outward K+ current, I(to), by 4-aminopyridine (4-AP) was studied in ferret right ventricular myoc ytes using the whole cell patch clamp technique. 4-AP reduces I(to) th rough a closed state blocking mechanism displaying ''reverse use-depen dent'' behavior that was inferred from: (a) development of tonic block at hyperpolarized potentials; (b) inhibition of development of tonic block at depolarized potentials; (c) appearance of ''crossover phenome na'' in which the peak current is delayed in the presence of 4-AP at d epolarized potentials; (d) relief of block at depolarized potentials w hich is concentration dependent and parallels steady-state inactivatio n for low 4-AP concentrations (V1/2 almost-equal-to -10 mV in 0.1 mM 4 -AP) and steady-state activation at higher concentrations (V1/2 = +7 m V in 1 mM 4-AP, +15 mV in 10 mM 4=AP); and (e) reassociation of 4-AP a t hyperpolarized potentials. No evidence for interaction of 4-AP with either the open or inactivated state of the I(to) channel was obtained from measurements of kinetics of recovery and deactivation in the pre sence of 0.5-1.0 mM 4-AP. At hyperpolarized potentials (-30 to -90 mV) 10 mM 4-AP associates slowly (time constants ranging from approximate ly 800 to 1,300 ms) with the closed states of the channel (apparent K( d) almost-equal-to 0.2 mM). From -90 to -20 mV the affinity of the I(t o) channel for 4-AP appears to be voltage insensitive; however, at dep olarized potentials (+20 to +100 mV) 4-AP dissociates with time consta nts ranging from approximately 350 to 150 ms. Consequently, the proper ties of 4-AP binding to the I(to) channel undergo a transition in the range of potentials over which channel activation and inactivation occ urs (-30 to +20 mV). We propose a closed state model of I(to) channel gating and 4-AP binding kinetics, in which 4-AP binds to three closed states. In this model 4-AP has a progressively lower affinity as the c hannel approaches the open state, but has no intrinsic voltage depende nce of binding.