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Department of Neurobiology, Harvard Medical School, Boston, MA 02115

Correspondence to Gary Yellen: gary_yellen{at}hms.harvard.edu

Voltage-dependent K⁺ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421–439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389–414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd²⁺ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the intracellular gate remains an effective barrier to the movement of potassium ions through the pore.

M. Kanevsky's present address is Department of Anesthesia, Stanford University School of Medicine, 300 Pasteur Drive, H3580, Stanford, CA 94305.

Abbreviations used in this paper: 4-AP, 4-aminopyridine; MTSET, trimethylaminoethyl methanethiosulfonate; MTSHE, hydroxyethyl methanethiosulfonate.

¹ The values of the rate constants for the open state reaction also agree reasonably well for channels with and without the LT mutations (1.2 x 10⁵ for 478C-LT vs. 0.8 x 10⁵ for 478C; the latter value from Liu et al., 1997; del Camino and Yellen, 2001).

² The 474C-ILT channel opens at less extreme depolarized voltages than the wild-type ILT channel, so we used the ILT version of the 474C channel (rather than the LT version used for 478C).

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