Gated Access to the Pore of a Voltage-Dependent K+ Channel

Abstract

Voltage-activated K+ channels are integral membrane proteins that open or close a K(+)-selective pore in response to changes in transmembrane voltage. Although the S4 region of these channels has been implicated as the voltage sensor, little is known about how opening and closing of the pore is accomplished. We explored the gating process by introducing cysteines at various positions thought to lie in or near the pore of the Shaker K+ channel, and by testing their ability to be chemically modified. We found a series of positions in the S6 transmembrane region that react rapidly with water-soluble thiol reagents in the open state but not the closed state. An open-channel blocker can protect several of these cysteines, showing that they lie in the ion-conducting pore. At two of these sites, Cd2+ ions bind to the cysteines without affecting the energetics of gating; at a third site, Cd2+ binding holds the channel open. The results suggest that these channels open and close by the movement of an intracellular gate, distinct from the selectivity filter, that regulates access to the pore.

Gating at the selectivity filter in cyclic nucleotide-gated channels

“…For example, studies using intracellularly applied molecules that block the permeation pathway of CNG channels, such as divalent ions (18), tetracaine (19,20), or quaternary ammonium ions (21), have shown that blockade is not state-dependent, as if these molecules can access the pore in both open and closed channels, which is in stark contrast with the blockade properties observed in K V channels (10,11,14,(22)(23)(24). In addition, experiments examining the state dependence of cysteine modification by intracellular application of methanethiosulfonate (MTS) reagents have failed to show dramatic differences between open and closed states in the inner-vestibule region (21,25,26), results that are inconsistent with an intracellular gate in TM6, as shown in K V channels (12,15).Several studies indicate that the pore region of CNG channels plays a role in gating. For example, accessibility of cysteine reagents applied from the intracellular (27, 28) and the extracellular (27-29) side of the channel to cysteines substituted along the entire P region have shown that modification of some residues in this region perturbed normal gating by cGMP.…”

“…A large body of evidence, using a variety of approaches, has established that K V channels open and close their permeation pathway at the intracellular end of the pore (10)(11)(12)(13)(14)(15)(16)(17). Attempts to extend those ideas to CNG channels have encountered some resistance.…”

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K + channel

“…Genetic analysis, coupled with biophysical analysis, of TOK1 has assisted in the identification of what we call the PP region, the cytoplasmic end of the P-region-following membrane domain, which has been found to be intimately involved with gating of cation channels (1,2,3). Our analyses have also pointed to the existence of functional domains that, on first glance, appeared unique to TOK1, including a filter-specific gating ( Fig.…”

“…Mutations exclusively in the ''PP'' region (the end of M6 and M8 in the case of TOK1) specifically disrupt the C states (1) suggesting that they result from a constriction of the inner mouth of the pore, akin to deactivation-type gating in other cation channels (2,3,23,24). We have proposed that the K ext ϩ ͞Vm dependence of C could simply result from the open noncollapsed filter preventing inner pore gating transitions (4).…”

Grafting voltage and pharmacological sensitivity in potassium channels

“…Notably, the study on KvLm channels revealed that pore only module can populate at open state, suggesting that the pore of Kv channels may also reside in between closed-activated and open state [16]. The gating of the Kv channel pore involves both the lower bundle-crossing gate at the cytoplasmic entrance to the channel and the upper gate at the selectivity filter close to the outer mouth of the channel [54,55]. By contrast, the gating of K2P channels was thought to occur through their upper gate [56][57][58][59].…”