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Phosphatidylinositol (4,5)-bisphosphate (PIP(2)) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP(2) on the open state stabilization. A similar effect of PIP(2) on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K(+) channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP(2) effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP(2) exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations.

The co-assembly of KCNQ1 with KCNE1 produces I(KS), a K(+) current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K(+) channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.

The highly conserved fourth transmembrane segment (S4) is the primary voltage sensor of the voltage-dependent channel and would move outward upon membrane depolarization. S4 comprises repetitive amino acid triads, each containing one basic (presumably charged and voltage-sensing) followed by two hydrophobic residues. We showed that the triad organization is functionally extended into the S3-4 linker right external to S4 in Shaker K(+) channels. The arginine (and lysine) substitutes for the third and the sixth residues (Ala-359 and Met-356, respectively) external to the outmost basic residue (Arg-362) in S4 dramatically and additively stabilize S4 in the resting conformation. Also, Leu-361 and Leu-358 play a very similar role in stabilization of S4 in the resting position, presumably by their hydrophobic side chains. Moreover, the double mutation A359R/E283A leads to a partially extruded position of S4 and consequently prominent closed-state inactivation, suggesting that Glu-283 in S2 may coordinate with the arginines in the extruded S4 upon depolarization. We conclude that the triad organization extends into the S3-4 linker for about six amino acids in terms of their microenvironment. These approximately six residues should retain the same helical structure as S4, and their microenvironment serves as part of the "gating canal" accommodating the extruding S4. Upon depolarization, S4 most likely moves initially as a sliding helix and follows the path that is set by the approximately six residues in the S3-4 linker in the resting state, whereas further S4 translocation could be more like, for example, a paddle, without orderly coordination from the contiguous surroundings.

The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K(+)-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K(+)-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling.

Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca(2+) and K(+) channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K(+) channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H(2)O(2). We show that H(2)O(2) rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H(2)O(2) and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H(2)O(2)-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys(168) located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H(2)O(2). The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys(168) as a critical target for H(2)O(2), and implicate ROS-mediated control of the K(+) channel in regulating mineral nutrient partitioning within the plant.

Voltage-gated ion channels are controlled by the membrane potential, which is sensed by peripheral, positively charged voltage sensors. The movement of the charged residues in the voltage sensor may be detected as gating currents. In Shaker K(+) channels, the gating currents are asymmetric; although the on-gating currents are fast, the off-gating currents contain a slow component. This slow component is caused by a stabilization of the activated state of the voltage sensor and has been suggested to be linked to ion permeation or C-type inactivation. The molecular determinants responsible for the stabilization, however, remain unknown. Here, we identified an interaction between Arg-394, Glu-395, and Leu-398 on the C termini of the S4-S5 linker and Tyr-485 on the S6 of the neighboring subunit, which is responsible for the development of the slow off-gating component. Mutation of residues involved in this intersubunit interaction modulated the strength of the associated interaction. Impairment of the interaction still led to pore opening but did not exhibit slow gating kinetics. Development of this interaction occurs under physiological ion conduction and is correlated with pore opening. We, thus, suggest that the above residues stabilize the channel in the open state.

The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.