Following voltage-dependent activation, Drosophila Shaker K+ channels enter a nonconducting, inactivated state. This process has been proposed to occur by a “ball-and-chain” mechanism, in which the N-terminus of the protein behaves like a blocker tethered to the cytoplasmic side of the channel and directly occludes the pore to cause inactivation. To complement the ample evidence for the involvement of the N-terminus, we sought evidence that it blocks the pore directly. We found that inactivation exhibits several distinctive properties of pore blockade. First, recovery was speeded by increased external K+ concentrations, just as blockade can be relieved by trans-permeant ions. Second, singlechannel experiments show that the channel reopens from the inactivated state upon repolarization. These openings were usually required for recovery, as though the blocking particle must exit the pore before the channel can close. introduction

Voltage-dependent K+ channels are found in many Types of excitable and nonexcitable cells. Theyconsti-: ute a diverse class of channels that may be distinguished by their differences in gating, conductance,* md pharmacology. These channels open upon depoiarization and enter a long-lasting, nonconductive state (the inactivated state), from which they cannot, e activated by depolarization. Among the different nembers of this class of channels, the rates of inacti-*/ation vary from milliseconds to seconds. Physiologie: ally, this variation affects the generation, propagaion, and integration of electrical signals in cells. The transient K+ channels encoded by the Drosophla Shaker gene are among the most rapidly inactivatng type of K+ channel. Like voltage-activated Na+: hannels, they inactivate within a few milliseconds. ifter opening. Recent experiments on Shaker K+ chanlels support a “ball-and-chain” mechanism for their ‘ast inactivation, like the mechanism originally pro-> osed byBezaniIlaandArmstrong (1977) for Na+ chanlel inactivation. Both types of channels inactivate lfter opening, and the inactivation can be abolished 3)~ mild intracellular protease treatment (Armstrong? t al., 1973; Hoshi et al., 1990). Protease treatment is: hought to remove an inactivation particle that is nornally tethered to the cytoplasmic side of the channel. n the ball-and-chain model, inactivation is caused by