MODULATION OF 4-AP BLOCK OF A MAMMALIAN A-TYPE K-CHANNEL CLONE BY CHANNEL GATING AND MEMBRANE VOLTAGE

We examined the state-, voltage-, and time dependences of interaction between 4-AP and a mammalian A-type K channel clone (rKv1.4) expressed in Xenopus oocytes using whole-cell and single-channel recordings. 4- AP blocked rKv1.4 from the cytoplasmic side of the membrane. The devel opment of block required channel opening. Block was potentiated by rem oving the fast inactivation gate of the channel (deletion mutant terme d ''Del A''), A short-pulse train that activated rKv1.4 without inacti vation induced more block by 4-AP than a long pulse that activated and then inactivated the channel. These observations suggest that both ac tivation and inactivation gates limit the binding of 4-AP to the chann el. Unblock of 4-AP also occurred during channel opening, because unbl ock required depolarization and was accelerated by more frequent or lo nger depolarization pulses (use-dependent unblock). Analysis of the co ncentration dependence of rate of block development indicated that 4-A P blocked rKv1.4 with slow kinetics (at -20 mV, binding and unbinding rate constants were 3.2 mM(-1) s(-1) and 4.3 s(-1)). This was consiste nt with single-channel recordings: 4-AP induced little or no changes i n the fast kinetics of opening and closing within bursts, but shortene d the mean burst duration and, more importantly, reduced the probabili ty of channel opening by depolarization. Depolarization might decrease the affinity of 4-AP binding site in the open channel, because strong er depolarization reduced the degree of steady-state block by 4-AP. Fu rthermore, after 4-AP block had been established at a depolarized hold ing voltage, further depolarization induced a time-dependent unblock. Our data suggest that 4-AP binds to and unbinds from open rKv1.4 chann els with slow kinetics, with the binding site accessibility controlled by the channel gating apparatus and binding site affinity modulated b y membrane voltage.