期刊
JOURNAL OF GENERAL PHYSIOLOGY
卷 126, 期 5, 页码 419-428出版社
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.200509385
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- NINDS NIH HHS [R01 NS029693, NS 29693] Funding Source: Medline
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.
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