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

Abstract

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.

Figure 1. Identification of pore-facing and non–pore-facing amino acids of the Psora-4 binding site

(a) Alignment of the pore helix and pore forming S6 segment of Kv channels. The classical drug-binding site is highlighted in gray. SF, selectivity filter. (b) Block of different Kv channels by 500 nM Psora-4, analyzed at +40 mV. Inset shows the structure of Psora-4. (c) Dose-response relationship for Kv1.5 channels. n = 7–20 per concentration. (d) Kv1.5 currents under control conditions and repetitive pulses directly after a 12-min pulse-free period in the presence of 500 nM Psora-4 (n = 7). The upper panel illustrates the voltage protocol, and the inset shows representative measurements. (e) Wild-type (WT) and mutant channel currents before and after incubation with 500 nM Psora-4. (f) Alanine scan of the S6 using 500 nM Psora-4, analyzed at +40 mV. SF, selectivity filter. (g) Localization of pore-facing and non–pore-facing residues in an open-state Kv1.5 homology model. (h,i) Alanine scan of the S4, S4-S5 linker (h) and the S5 segment (i). In e–i, red and blue coloring refers to pore-facing and non–pore-facing residues, respectively. Data are represented throughout the figure as mean ± s.e.m. The number of experiments (n) are indicated as small insets within the bars. ^***P < 0.001. NE, not expressing.

Figure 2. Psora-4 binding site in the central cavity and side pockets

(a,b) Kv1.5 open-state homology model with Psora-4 molecules shown in orange, viewed from the extracellular face (a) and from the side (b). (c,d) Docking of Psora-4 in the central cavity (c) and the side pockets (d). The residues identified by mutagenesis are shown as colored sticks (red, classical pore drug-binding site; blue, side-pocket binding site). The binding orientation for Psora-4, as predicted by GOLD, is shown in orange.

Figure 3. Introducing Psora-4 affinity into Kv2.1 channels by creating a Kv1-like side pocket

(a) Alignment of Kv1.5, Kv2.1, the four-fold mutant Kv2.1 SP and the six-fold mutant Kv2.1 SP+CC. The Psora-4 binding site is highlighted in bold. Asterisks indicate conserved residues of the binding site. The Kv2.1 mutations at the nonconserved sites are marked by arrows (red, central cavity; blue, side pocket). (b) Psora-4 effect on Kv1.5 and Kv2.1 channels expressed in oocytes. WT, wild type. (c) Dose-response curves of Kv1.5, Kv2.1, Kv2.1 SP and Kv2.1 SP+CC for Psora-4; n = 4–20 per concentration. (d) Examples of the dose-response relationships from binding-site mutant channels of each drug-binding domain; n = 3–22 per concentration. (e) Open pore homology model of Kv1.5, including the S6 and the pore loops of four subunits. Psora-4 molecules are shown in orange. One Psora-4 molecule is bound to the central cavity, and additional Psora-4 molecules are illustrated in two opposing side pockets. The proline-valine-proline (PVP) sequences are highlighted as yellow spheres. (f) Open pore homology model of Kv1.5 with Ile508 (red) facing the pore and Ile502 (light gray) facing the lateral fenestrations. SF, selectivity filter. Data are represented throughout the figure as mean ± s.e.m.

Figure 4. Psora-4 in the side pocket interacts with the pore helix

(a) MDS result where one Psora-4 molecule bound in the side pockets is reaching the pore helix. Psora-4 is shown in orange. (b) Mutant channel currents before and after incubation with 500 nM Psora-4. (c) Inhibition of pore helix mutants by 500 nM Psora-4, analyzed at +40 mV. WT, wild type. (d) Inhibition of pore helix mutant channels by 250 μM bupivacaine. In c and d, the number of experiments (n) are indicated within the bars. (e) Inhibition of A503L mutant by 500 nM Psora-4. (f) Fold changes in IC₅₀ introducing different residues at position 503; n = 3–13 per concentration. (g) Residues of the pore helix and A503 (S6) that interact with Psora-4 are highlighted as blue spheres. (h) Large residues at position 503 (A503L) might interfere with Psora-4 access to the pore helix. Data are represented throughout the figure as mean ± s.e.m. ^***P < 0.001. NE, not expressing.

Figure 5. Psora-4 blocking mechanism

(a) Superimposed Kv1.5 gating currents recorded at +60 mV before (control) and after addition of 500 nM Psora-4 for 15 min without pulsing (Psora-4) and after 20 pulses to +60 mV (Psora-4, pulses). Middle and right panels illustrate the Q_ON and Q_OFF voltage relationships (n = 5). (b–h) Inside-out macropatch recordings from Xenopus oocytes. (b) Inhibition of Kv1.5 by low Psora-4 doses (left) and block by different Psora-4 concentrations (right). (c) Time course of wash-in and wash-out of 100 nM Psora-4. (d) Time course of inhibition and reversibility of block for drug-binding site mutations in the central cavity (CC, red) and side pocket (SP, blue). After the control pulse (not shown), patches were incubated with 100 nM Psora-4 for 3 min. The first and second pulse in the presence of Psora-4 are illustrated. Kinetics of block were analyzed with a monoexponential fit (see Online Methods for batch-dependent analyses). (e) The kinetics of block by Psora-4 in the absence (left; n = 4) and presence of TEA (right; n = 3). τ_P4 is the time constant of inhibition by Psora-4. (f) Inside-out patch clamp recordings of Psora-4 alone or after pre-application of either TEA (g) or TPA (h). In g and h, the models depict the occupancy of the different binding sites by Psora-4 (P4), the competition of the binding in the central cavity and the proposed effects on the selectivity filter. The triangular shape of TEA indicates the stabilization of the selectivity filter. The arrows mark the stable pore collapse. Data are represented as mean ± s.e.m.