REVERSE USE DEPENDENCE OF KV4.2 BLOCKADE BY 4-AMINOPYRIDINE

4-Aminopyridine (4AP) can block various K channels with different stat e dependences; block occurs in the activated state or in the closed st ate. The use of K channel clones to study the mechanism and structural determinants responsible for the state dependence of 4AP actions has been hampered by the fact that, for all the K channel clones examined so far, 4AP binding and unbinding occur mainly in the activated state. We report here that 4AP binding to a fast inactivating K channel enco ded by Kv4.2 in Xenopus oocytes occurred exclusively in the closed sta te. The binding rate was slow and independent of membrane voltage in t he range from -80 to -120 mV. The binding rate was linearly related to 4AP concentration, yielding apparent binding and unbinding rate const ants of 0.012 mM(-1) s(-1) and 0.062 s(-1), respectively. 4AP dissocia tion from Kv4.2 occurred in two processes, a slow process in the close d state (in a voltage range from -70 to -40 mV) and a fast process in the activated state, which suggested that the closure of the activatio n gate of Kv4.2 did not prevent the entry or exit of 4AP molecules but slowed these processes. 4AP slowed the rate of Kv4.2 decay during dep olarization, consistent with the notion that channel inactivation occu rred only after 4AP dissociation. Inactivating Kv4.2 channels prevente d 4AP binding. Therefore, 4AP binding and Kv4.2 inactivation were mutu ally exclusive. This, in conjunction with the observation that 4AP blo cked Kv4.2 channels from the intracellular side of the cell membrane, suggests that the 4AP binding site is on the cytoplasmic surface of th e Kv4.2 channel at, or adjacent to, the domains involved in channel in activation. The distinct features of 4AP actions on the time course of transient outward current in human ventricular myocytes suggest that Kv4.2-like subunits are important in the formation of these channels i n human heart.