Identification of a critical binding site for local anaesthetics in the side pockets of Kv 1 channels.

BACKGROUND AND PURPOSE: Local anaesthetics block sodium and a variety of potassium channels. Although previous studies identified a residue in the pore signature sequence together with three residues in the S6 segment as a putative binding site, the precise molecular basis of inhibition of Kv channels by local anaesthetics remained unknown. Crystal structures of Kv channels predict that some of these residues point away from the central cavity and face into a drug binding site called side pockets. Thus, the question arises whether the binding site of local anaesthetics is exclusively located in the central cavity or also involves the side pockets. EXPERIMENTAL APPROACH: A systematic functional alanine mutagenesis approach, scanning 58 mutants, together with in silico docking experiments and molecular dynamics simulations was utilized to elucidate the binding site of bupivacaine and ropivacaine. KEY RESULTS: Inhibition of Kv 1.5 channels by local anaesthetics requires binding to the central cavity and the side pockets, and the latter requires interactions with residues of the S5 and the back of the S6 segments. Mutations in the side pockets remove stereoselectivity of inhibition of Kv 1.5 channels by bupivacaine. Although binding to the side pockets is conserved for different local anaesthetics, the binding mode in the central cavity and the side pockets shows considerable variations. CONCLUSION AND IMPLICATIONS: Local anaesthetics bind to the central cavity and the side pockets, which provide a crucial key to the molecular understanding of their Kv channel affinity and stereoselectivity, as well as their spectrum of side effects.

Side pockets provide the basis for a new mechanism of Kv channel-specific inhibition.

Most known small-molecule inhibitors of voltage-gated ion channels have poor subtype specificity because they interact with a highly conserved binding site in the central cavity. Using alanine-scanning mutagenesis, electrophysiological recordings and molecular modeling, we have identified a new drug-binding site in Kv1.x channels. We report that Psora-4 can discriminate between related Kv channel subtypes because, in addition to binding the central pore cavity, it binds a second, less conserved site located in side pockets formed by the backsides of S5 and S6, the S4-S5 linker, part of the voltage sensor and the pore helix. Simultaneous drug occupation of both binding sites results in an extremely stable nonconducting state that confers high affinity, cooperativity, use-dependence and selectivity to Psora-4 inhibition of Kv1.x channels. This new mechanism of inhibition represents a molecular basis for the development of a new class of allosteric and selective voltage-gated channel inhibitors.

A leucine zipper motif essential for gating of hyperpolarization-activated channels.

BACKGROUND: It is poorly understood how hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) function. RESULTS: We have identified a leucine zipper in the S5 segment of HCNs, regulating hyperpolarization-activated and instantaneous current components. CONCLUSION: The leucine zipper is essential for HCN channel gating. SIGNIFICANCE: The identification and functional characterization of the leucine zipper is an important step toward the understanding of HCN channel function. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are pacemakers in cardiac myocytes and neurons. Although their membrane topology closely resembles that of voltage-gated K(+) channels, the mechanism of their unique gating behavior in response to hyperpolarization is still poorly understood. We have identified a highly conserved leucine zipper motif in the S5 segment of HCN family members. In order to study the role of this motif for channel function, the leucine residues of the zipper were individually mutated to alanine, arginine, or glutamine residues. Leucine zipper mutants traffic to the plasma membrane, but the channels lose their sensitivity to open upon hyperpolarization. Thus, our data indicate that the leucine zipper is an important molecular determinant for hyperpolarization-activated channel gating. Residues of the leucine zipper interact with the adjacent S6 segment of the channel. This interaction is essential for voltage-dependent gating of the channel. The lower part of the leucine zipper, at the intracellular mouth of the channel, is important for stabilizing the closed state. Mutations at these sites increase current amplitudes or result in channels with deficient closing and increased min-P(o). Our data are further supported by homology models of the open and closed state of the HCN2 channel pore. Thus, we conclude that the leucine zipper of HCN channels is a major determinant for hyperpolarization-activated channel gating.

RNA editing modulates the binding of drugs and highly unsaturated fatty acids to the open pore of Kv potassium channels.

The time course of inactivation of voltage-activated potassium (Kv) channels is an important determinant of the firing rate of neurons. In many Kv channels highly unsaturated lipids as arachidonic acid, docosahexaenoic acid and anandamide can induce fast inactivation. We found that these lipids interact with hydrophobic residues lining the inner cavity of the pore. We analysed the effects of these lipids on Kv1.1 current kinetics and their competition with intracellular tetraethylammonium and Kvbeta subunits. Our data suggest that inactivation most likely represents occlusion of the permeation pathway, similar to drugs that produce 'open-channel block'. Open-channel block by drugs and lipids was strongly reduced in Kv1.1 channels whose amino acid sequence was altered by RNA editing in the pore cavity, and in Kv1.x heteromeric channels containing edited Kv1.1 subunits. We show that differential editing of Kv1.1 channels in different regions of the brain can profoundly alter the pharmacology of Kv1.x channels. Our findings provide a mechanistic understanding of lipid-induced inactivation and establish RNA editing as a mechanism to induce drug and lipid resistance in Kv channels.

Kvbeta1.3 reduces the degree of stereoselective bupivacaine block of Kv1.5 channels.

BACKGROUND: Kvbeta1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kvbeta1.3 competes with them for a binding site at Kv1.5 channels. METHODS: Currents generated by the activation of Kv1.5 and Kv1.5 + Kvbeta1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques. RESULTS: Block of Kv1.5, but not that produced on Kv1.5 + Kvbeta1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(-)-bupivacaine blocked Kv1.5 with IC50 4.4 +/- 0.5 microM (n = 15) and 39.8 +/- 8.2 microM (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 +/- 2.2 microM) and twofold for S(-)-bupivacaine (71.9 +/- 11.5 microM) in Kv1.5 + Kvbeta1.3 channels. Therefore, the degree of stereoselectivity (theta) decreased from 9 to 4 in the presence of Kvbeta1.3. The decrease in potency to block Kv1.5 + Kvbeta1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kvbeta1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kvbeta1.3 binding) but not for L510A (part of Kvbeta1.3 binding site). CONCLUSIONS: The degree of stereoselective block of Kv1.5 decreases from 9 to 4 when Kvbeta1.3 is present. L510 is determinant for the modulation of bupivacaine block, because it is the only residue of the S6 segment that binds to both bupivacaine and Kvbeta1.3. These findings support an overlapping binding site for drugs and Kvbeta1.3.