The recent crystallization of a voltage-gated K ؉ channel has given insight into the structure of these channels but has not resolved the issues of the location and the operation of the gate. The conserved PXP motif in the S6 segment of Shaker channels has been proposed to contribute to the intracellular gating structure. To investigate the role of this motif in the destabilization of the ␣-helix, both prolines were replaced to promote an ␣-helix (alanine) or to allow a flexible configuration (glycine). These substitutions were nonfunctional or resulted in drastically altered channel gating, highlighting an important role of these prolines. Combining these mutations with a proline substitution scan demonstrated that proline residues in the midsection of S6 are required for functionality, but not necessarily at the positions conserved throughout evolution. These results indicate that the destabilization or bending of the S6 ␣-helix caused by the PXP motif apparently creates a flexible "hinge" that allows movement of the lower S6 segment during channel gating and opening.

The selectivity filter and the activation gate in potassium channels are functionally and structurally coupled. An allosteric coupling underlies C-type inactivation coupled to activation gating in this ion-channel family (i.e., opening of the activation gate triggers the collapse of the channel's selectivity filter). We have identified the second Threonine residue within the TTVGYGD signature sequence of K channels as a crucial residue for this allosteric communication. A Threonine to Alanine substitution at this position was studied in three representative members of the K-channel family. Interestingly, all of the mutant channels exhibited lack of C-type inactivation gating and an inversion of their allosteric coupling (i.e., closing of the activation gate collapses the channel's selectivity filter). A state-dependent crystallographic study of KcsA-T75A proves that, on activation, the selectivity filter transitions from a nonconductive and deep C-type inactivated conformation to a conductive one. Finally, we provide a crystallographic demonstration that closed-state inactivation can be achieved by the structural collapse of the channel's selectivity filter.

Voltage-dependent K + channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6 T ) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6 T . Exchanging both L45 and S6 T transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6 T in hKv1.5 further shows that S6 T needs to be ␣ -helical and forms a " crevice " in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6 T crevice, whereas other residues in S6 T and L45 that are not involved in the interaction maintain the correct structure of the coupling.

Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K+ permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K+ conduction through constriction of the K+ selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the subsequent membrane repolarization. Measurements of deactivating gating currents (reporting VSD movement) and ionic currents (BC gate status) showed that the kinetics of both slowed down in two distinct phases with increasing duration of the depolarizing prepulse. The biphasic slowing in VSD deactivation and BC gate closure was strongly correlated in time and magnitude. Simultaneous recordings of ionic currents and fluorescence from a probe tracking VSD movement in Shaker directly demonstrated that both processes were synchronized. Whereas the first slowing originates from a stabilization imposed by BC gate opening, the subsequent slowing reflects the rearrangement of the VSD toward its relaxed state (relaxation). The VSD relaxation was observed in the Ciona intestinalis voltage-sensitive phosphatase and in its isolated VSD. Collectively, our results show that the VSD relaxation is not kinetically related to C-type inactivation and is an intrinsic property of the VSD. We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history.

Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4 Voltage-gated ion channels are ubiquitously expressed in human tissues where they play diverse physiological functions such as generation and modulation of the electrical activity in excitable tissues, myocyte contraction, modulation of neurotransmitter and hormone release, and electrolyte transport in epithelia. Crystallization and x-ray diffraction of both a prokaryote and a mammalian voltage-gated potassium (Kv) channel provided a lot of information on the structure of Kv channels (1-3). Even though these data initiated controversies on the dynamics of the voltage sensor, S4 (4), they provided a new template for investigations on Kv channel molecular characteristics. For example, the structure of the Kv pore domain (S5-S6) turned out to be similar to the pore domain of two-transmembrane domain potassium channels like KcsA (5) and KirBac1.1 (6). Besides structure, the crystallographic analyses of KvAP and Kv1.2 gave insights on the dynamics of channel voltage dependence. Notably, the crystal structure of Kv1.2 is believed to represent the channel in an open state (3), and in this conformation, the S4-S5 linker (S4S5 L ) 7 is interacting with the S6 C terminus (S6 T ). The authors (3) used homology modeling to infer a closed state structure from the open state structure. In the closed state, the model shows that S4S5 L and S6 T are also in contact. Those results pointed to a mechanism by which S4S5 L are permanently linked to S6, and this link is critical in translating the voltage sensor movement into gate opening or closure (3).In a few other channels, the S4S5 L /S6 T interaction seems rather state-dependent. A second-site suppressor yeast screen in the hyperpolarization-activated channel KAT1 suggested that S4S5 L and S6 T are interacting only in the channel open state (7). In another hyperpolarization-activated channel,

Gambierol is a marine polycyclic ether toxin belonging to the group of ciguatera toxins. It does not activate voltage-gated sodium channels (VGSCs) but inhibits Kv1 potassium channels by an unknown mechanism. While testing whether Kv2, Kv3, and Kv4 channels also serve as targets, we found that Kv3.1 was inhibited with an IC 50 of 1.2 ؎ 0.2 nM, whereas Kv2 and Kv4 channels were insensitive to 1 M gambierol. Onset of block was similar from either side of the membrane, and gambierol did not compete with internal cavity blockers. The inhibition did not require channel opening and could not be reversed by strong depolarization. Using chimeric Kv3.1-Kv2.1 constructs, the toxin sensitivity was traced to S6, in which T427 was identified as a key determinant. In Kv3.1 homology models, T427 and other molecular determinants (L348, F351) reside in a space between S5 and S6 outside the permeation pathway. In conclusion, we propose that gambierol acts as a gating modifier that binds to the lipid-exposed surface of the pore domain, thereby stabilizing the closed state. This site may be the topological equivalent of the neurotoxin site 5 of VGSCs. Further elucidation of this previously undescribed binding site may explain why most ciguatoxins activate VGSCs, whereas others inhibit voltage-dependent potassium (Kv) channels. This previously undescribed Kv neurotoxin site may have wide implications not only for our understanding of channel function at the molecular level but for future development of drugs to alleviate ciguatera poisoning or to modulate electrical excitability in general.ciguatera ͉ neurotoxin site 5 ͉ polycyclic ether toxin ͉ potassium channels ͉ Kv3.1

The charge versus voltage relation of voltage-sensor domains shifts in the voltage axis depending on the initial voltage. Here we show that in nonconducting W434F Shaker K(+) channels, a large portion of this charge-voltage shift is apparent due to a dramatic slowing of the deactivation gating currents, Ig(D) (with τ up to 80 ms), which develops with a time course of ∼1.8 s. This slowing in Ig(D) adds up to the slowing due to pore opening and is absent in the presence of 4-aminopyridine, a compound that prevents the last gating step that leads to pore opening. A remaining 10-15 mV negative shift in the voltage dependence of both the kinetics and the charge movement persists independently of the depolarizing prepulse duration and remains in the presence of 4-aminopyridine, suggesting the existence of an intrinsic offset in the local electric field seen by activated channels. We propose a new (to our knowledge) kinetic model that accounts for these observations.

In vivo, KCNQ1 ␣-subunits associate with the ␤-subunit KCNE1 to generate the slowly activating cardiac potassium current (I Ks ). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1-S6) with the S1-S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5 L ) and the C-terminal end of S6 (S6 T ). Previously we reported that substitutions for Leu 353 in S6 T resulted in channels that failed to close completely. Closure could be incomplete because Leu 353 itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5 L in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5 L . The residues with a "high impact" on channel gating (when mutated) clustered on one side of the S4S5 L ␣-helix. Hence, this side of S4S5 L most likely contributes to the electromechanical coupling and finds its residue counterparts in S6 T . Accordingly, substitutions for Val 254 resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu 353 in S6 T . Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.KCNQ1 (KvLQT1) ␣-subunits tetramerize to create a voltage-gated K ϩ (Kv) channel. Like other Kv channels, each ␣-subunit contains six membrane spanning segments (S1-S6) with a pore loop between the fifth and sixth segment that forms the selectivity filter. The co-assembly with KCNE1 (minK) ␤-subunits generates the channel complex that underlies the native I Ks in the heart (1, 2). A fundamental property of all Kv channels is their ability to detect a change in membrane potential (V m ) and to respond to this change by opening or closing their activation gate that seals off the ion permeation pathway in a closed configuration (3, 4). The channel activation gate is located in the C-terminal end of the S6 segment (S6 T ), 3 whereas the voltage-sensing domain (VSD) is formed by the S1-S4 segments with the charged S4 as the main component (5). Substitution of the S4 charges in KCNQ1 perturbed channel gating (6, 7) and gating currents that originate from the redistribution of these S4 charges have recently been recorded for the KCNQ family of Kv channels (8). These data indicate that as in Shaker-type channels, the S1-S4 segment in KCNQ1 forms the channel VSD that reorients upon a change in membrane potential. This VSD reorientation is then translated into channel gate opening or closure through an electromechanical coupling. This coupling mechanism remains poorly defined but sev...