Gating currents from Kv7 channels carrying neuronal hyperexcitability mutations in the voltage-sensing domain

Abstract

Changes in voltage-dependent gating represent a common pathogenetic mechanism for genetically inherited channelopathies, such as benign familial neonatal seizures or peripheral nerve hyperexcitability caused by mutations in neuronal K(v)7.2 channels. Mutation-induced changes in channel voltage dependence are most often inferred from macroscopic current measurements, a technique unable to provide a detailed assessment of the structural rearrangements underlying channel gating behavior; by contrast, gating currents directly measure voltage-sensor displacement during voltage-dependent gating. In this work, we describe macroscopic and gating current measurements, together with molecular modeling and molecular-dynamics simulations, from channels carrying mutations responsible for benign familial neonatal seizures and/or peripheral nerve hyperexcitability; K(v)7.4 channels, highly related to K(v)7.2 channels both functionally and structurally, were used for these experiments. The data obtained showed that mutations affecting charged residues located in the more distal portion of S(4) decrease the stability of the open state and the active voltage-sensing domain configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S(4) caused activation of gating-pore currents at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying disease-causing mutations at different voltage-sensing domain locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases.

Figure 1

Ionic currents from K_v7.2 and K_v7.4 channels and gating currents from K_v7.4 channels. (A) Schematic representation of a K_v subunit. (B) Sequence alignment of the S₄ segments of the indicated K_v subunits (http://www.ebi.ac.uk/Tools/emboss/align/); residues mutated in this study are indicated at the top. Gray boxes indicate positively charged residues (numbered 0–7). (C) Representative macroscopic currents recorded from K_v7.2 (left) or K_v7.4 (right) channels expressed in Xenopus oocytes, in response to the indicated voltage protocols. (D) Representative currents recorded from K_v7.2- (left) or K_v7.4-expressing (right) oocytes after blockade of the ionic currents, in response to the indicated voltage protocols.

Figure 2

Functional effects of the R2Q mutation in S₄ in K_v7.2 and K_v7.4 channels. (A) Representative ionic current traces recorded from oocytes expressing K_v7.2 R2Q (left) and K_v7.4 R2Q (right) channels, in response to the indicated voltage protocols. (B) Averaged values of peak macroscopic current amplitudes at +40 mV (n = 6–13). (C) Averaged values of peak Q_ON at +40 mV (n = 6–13).

Figure 3

Ionic and gating currents from K_v7.4 channels carrying mutations in the C-terminal region of S₄. (A) Representative ionic (left) and gating (right) current traces recorded from oocytes expressing wild-type, R6W, R6Q, and D1G channels, in response to the voltage protocols indicated. (B and C) G/V (B) and Q_ON/V (C) curves for the indicated channels. Continuous lines represent Boltzmann fits to the experimental data. (D) Time constants for Q_ON and ionic current activation at +40 mV (n = 4–9). (E) Time constants for Q_OFF and ionic current deactivation at −100 mV (n = 4–9).

Figure 4

Ionic and gating currents from K_v7.4 channels carrying mutations in the central portion of S₄. (A) Representative macroscopic current traces elicited from oocytes expressing R4Q or R4W channels recorded in response to the indicated voltage protocol. (B) G/V curves for wild-type, R4Q, and R4W mutant channels. Continuous lines represent Boltzmann fits to the experimental data. (C) Representative gating current traces elicited from oocytes expressing wild-type, R4Q, or R4W channels, as indicated, recorded in response to the indicated voltage protocol. (D) Magnitude of the residual currents (gating-pore currents) calculated, after ionic current blockade, at the end of the voltage pulses from wild-type, R4Q, and R4W channels (n = 5–10). (E) Correlation between Q_OFF and gating-pore current magnitudes (both at + 60 mV) for the indicated channels. Each data point is from a single oocyte, from at least three separate batches. The two continuous lines represent linear regression fits for the data of wild-type and R4Q channels.

Figure 5

Molecular modeling of K_v7.4 and K_v7.4 R4Q VSDs. (A) Full K_v7.4 VSD in open/relaxed-state conformation after a 10-ns all-atom simulation, with components colored transparent gray (S1), yellow (S₂), red (S₃), and blue (S₄). (B and C) The salt-bridge interaction between R4 and E136 in S2 (B) is shown impeded by the R4Q mutation (C); also shown are the interactions involving the F143 residue in S₂. Both B and C show the VSD configurations at the end of molecular dynamics simulations (10 ns); the full movies of the 10-ns simulations for both wild-type and R4Q VSDs are provided as Movies S1 and S2, respectively.