Status of the Intracellular Gate in the Activated-not-open State of Shaker K+ Channels

Abstract

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.

Mechanism of Accelerated Current Decay Caused by an Episodic Ataxia Type-1-Associated Mutant in a Potassium Channel Pore

“…Therefore, it's plausible that current decay with the V478A mutation may in fact be due to perturbation of the activation gate region, promoting bundle crossing closing during depolarization and preventing secondary S4 movement. Consistently, 4-AP, which is known to associate with greatest preference to the activated-not-open state of the channel (Armstrong and Loboda, 2001;del Camino et al, 2005), binds with an ϳ20 times greater potency to Shaker IR A359C V478A compared with Shaker IR A359C when subjected to repetitive pulse protocols. Shaker IR A359C V478A had an IC50 of 48 M in 4-AP, compared with Shaker IR A359C with an IC50 of 868 M (data not shown).…”

“…4-AP is known to enter the inner pore of open channels and bind within the inner vestibule, blocking the permeation pathway and "pulling" the activation gate closed (McCormack et al, 1994;Armstrong and Loboda, 2001). This has the effect of trapping 4-AP inside the activation gate and antagonizing the concerted opening transition, thereby maintaining the channel in the "activated-not-open" state (del Camino et al, 2005). As a consequence, C-type inactivation, which takes place primarily from the open state, is largely prevented.…”

Tetrameric assembly of KvLm K + channels with defined numbers of voltage sensors

“…In the Kv Shaker, activation entails at least five kinetic transitions observable as gating currents: Three early transitions that are voltage-dependent but noncooperative are followed by two late transitions (3)(4)(5). At the end of the activation pathway, each subunit is in an "activated-notopen" conformation (6,7) referring to the state of the sensor and pore, respectively. Although it is well established that each sensor moves independently during the early transitions in activation (8), the nature of the interactions between subunits in Kv channels underlying the transition from activated-not-open to open (the opening transition) remains unsettled (6,(9)(10)(11)(12).…”

“…Although it is well established that each sensor moves independently during the early transitions in activation (8), the nature of the interactions between subunits in Kv channels underlying the transition from activated-not-open to open (the opening transition) remains unsettled (6,(9)(10)(11)(12). Structurally, the late kinetic transitions are considered to arise from conformational changes in the S4-S5 linker (7,13), whereas the final opening transition (6, 7) entails a change in conformation of S6, which forms the bundle-crossing of the pore (activation gate).…”

“…This dissection is possible when the opening transition is delayed or blocked. In Shaker, this has been achieved by blocking with 4-aminopyridine (7,14,15) or by mutations, including a single mutation at the beginning of the linker between the sensor and the pore (L382V) (3) or a triple mutation (ILT) in the S4 segment of the sensor (6,10,16,17). Alternatively, the assembly of hetero-oligomers composed of wild-type (WT) and mutant Kv subunits was explored as a means to examine inter-subunit interactions during voltage-dependent activation (4,6,(9)(10)(11)(12)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”