Neutralization of a negative charge in the S1-S2 region of the KV7.2 (KCNQ2) channel affects voltage-dependent activation in neonatal epilepsy

Abstract

The voltage-gated potassium channels KV7.2 and KV7.3 (genes KCNQ2 and KCNQ3) constitute a major component of the M-current controlling the firing rate in many neurons. Mutations within these two channel subunits cause benign familial neonatal convulsions (BFNC). Here we identified a novel BFNC-causing mutation (E119G) in the S1-S2 region of KV7.2. Electrophysiological investigations in Xenopus oocytes using two-microelectrode voltage clamping revealed that the steady-state activation curves for E119G alone and its coexpressions with KV7.2 and/or KV7.3 wild-type (WT) channels were significantly shifted in the depolarizing direction compared to KV7.2 or KV7.2/KV7.3. These shifts reduced the relative current amplitudes for mutant channels particularly in the subthreshold range of an action potential (about 45% reduction at --50 mV for E119G compared to KV7.2, and 33% for E119G/KV7.3 compared to KV7.2/KV7.3 channels). Activation kinetics were significantly slowed for mutant channels. Our results indicate that small changes in channel gating at subthreshold voltages are sufficient to cause neonatal seizures and demonstrate the importance of the M-current for this voltage range. This was confirmed by a computer model predicting an increased burst duration for the mutation. On a molecular level, these results reveal a critical role in voltage sensing of the negatively charged E119 in S1-S2 of KV7.2, a region that-- according to molecular modelling - might interact with a positive charge in the S4 segment.

Figure 1. Pedigree, BFNC-causing mutations within the K_V7.2 subunit and evolutionary conservation of E119

A, pedigree with clinical and genetic status. +/m: individuals carrying the A→G356 (E119G) mutation. Unaffected individuals are displayed with open symbols, affected ones with filled symbols. B, schematic view of a K_V7.2 subunit depicting all mutations that have been described so far as white symbols (Borgatti et al. 2004; Lerche et al. 2005). E119G is marked by a black symbol. C, E119 is located within the S1–S2 loop of K_V7.2 and is conserved in the orthologous K_V7.2 protein of human, mouse and rat. Analogous amino acids are marked in bold. GenBank accession numbers from top are: AY889405, AF490773, AF087453, AF071491, AY114213, AF105216, AF249278.

Figure 2. Functional analysis of homomeric and heteromeric K_V7.2 WT and mutant channels

A, representative raw current traces for K_V7.2 WT and E119G mutated channels. Currents were elicited from a holding potential of −80 mV by depolarizations ranging from −80 to +20 mV in 10 mV steps, followed by a pulse to −30 mV to obtain tail currents. B, conductance–voltage curves were constructed by plotting the normalized tail current amplitude recorded at −30 mV against the membrane potential. Lines represent standard Boltzmann functions fitted to the data points. Parameters were as follows: K_V7.2 (n = 12): V_0.5=−40.7 ± 0.9 mV, k =−8.4 ± 0.3 mV; E119G (n = 18): V_0.5=−36.4 ± 0.6 mV, k =−8.2 ± 0.3 mV; E119G/K_V7.2 (n = 5): V_0.5=−33.9 ± 0.8 mV, k =−8.1 ± 0.2 mV (P < 0.001 for V_0.5 for both conditions with mutant channels versus WT). Inset: representative current traces at −50 mV, normalized to the maximal current amplitude at +10 mV, illustrating the difference of current amplitudes. C, time constants of activation (τ_act) for K_V7.2, E119G and the E119G/K_V7.2 coexpression were obtained by fitting a first order exponential function to the rising part of each current trace. Values for τ_act were plotted against voltage, revealing an about 10 mV rightward shift of the voltage dependence of activation of E119G (n = 20) in comparison to K_V7.2 (n = 21). Activation time constants were significantly different for potentials between −40 mV and 30 mV (−40 mV and 20 mV: P < 0.01; −30 mV to 10 mV: P < 0.001; 30 mV: P < 0.05). For the coexpression of E119G/K_V7.2 channels (n = 5), the difference from WT K_V7.2 channels reached statistical significance for potentials between −50 mV and 10 mV (−50 mV to −30 mV: P < 0.001; −20 mV and −10 mV: P < 0.01; 0 mV and 10 mV: P < 0.05). D, time constants of deactivation (τ_deact) of K_V7.2 (n = 9) and E119G (n = 11) were evaluated by fitting a first order exponential function to the tail current decay at different potentials after a 1.5 s lasting depolarizing pulse to +50. Values for τ_deact were plotted against voltage and did not show a significant difference for potentials ranging between −140 mV and −100 mV. All data are shown as means ± s.e.m.

Figure 3. Functional analysis of heteromeric K_V7.2/K_V7.3 WT and mutant channels

A, conductance–voltage curves as described in Fig. 2B for coexpressions of K_V7.2/K_V7.3, E119G/K_V7.3 and E119G/K_V7.2/K_V7.3 channels. cRNA was injected in either a 1 : 1 or a 1 : 1 : 2 ratio. Lines represent standard Boltzmann functions fitted to the data points. Parameters were as follows: K_V7.2/K_V7.3 (n = 12): V_0.5=−39.4 ± 0.7 mV, k =−9.3 ± 0.5 mV; E119G/K_V7.3 (n = 8): V_0.5=−35.7 ± 0.7 mV (P < 0.01), k =−9.6 ± 0.9 mV; E119G/K_V7.2/K_V7.3 (n = 10): V_0.5=−37.4 ± 0.5 mV (P < 0.05), k =−9.1 ± 0.5 mV. B, time constants of activation (τ_act) for K_V7.2/K_V7.3 (n = 12), E119G/K_V7.3 (n = 9) and E119G/K_V7.2/K_V7.3 (n = 12) channels were obtained as described in the legend to Fig. 2. Activation kinetics were slowed for most of the potentials for E119G/K_V7.3 (−10 mV to 10 mV: P < 0.05; 20 mV to 60 mV: P < 0.01) and for E119G/K_V7.2/K_V7.3 (−50 mV: P < 0.05; −30 mV to 10 mV: P < 0.01; 20 mV to 40 mV: P < 0.001; 50 mV and 60 mV: P < 0.0001) in comparison to K_V7.2/K_V7.3 channels. C, time constants of deactivation (τ_deact) of K_V7.2/K_V7.3 (n = 5) and E119G/K_V7.3 (n = 6) were evaluated as described in the legend to Fig. 2 and were not significantly different. All data are shown as means ± s.e.m.

Figure 4. Comparison of maximal current amplitudes

Maximal current amplitudes were analysed after a depolarization lasting 2 s to 0 mV from a holding potential of −80 mV. To pool recordings from different experiments, amplitudes were normalized to the mean value of the amplitudes of K_V7.2 or K_V7.2/K_V7.3 that were obtained after simultaneous injections and recordings in parallel to the other clones. Co-expressions of the mutation with K_V7.2 or K_V7.3 were obtained by injection of a constant total amount of RNA in a 1 : 1 or 1 : 1 : 2 ratio in Xenopus oocytes. Two to three different batches of oocytes were injected. Normalized amplitudes ranged between 0.4 and 1.8 for K_V7.2 (n = 21) and between 0.1 and 1.7 for the E119G mutation (n = 20) revealing a broad overlap of data points. Pooled data from all batches exhibited a 34% decrease of the mean value of current amplitudes compared to the WT (P < 0.01). However, a reduction of the current amplitude could not be confirmed for either heteromeric E119G/K_V7.2 (n = 5) compared to K_V7.2, or E119G/K_V7.3 (n = 9) and E119G/K_V7.2/K_V7.3 (n = 6) compared to K_V7.2/K_V7.3 channels (n = 12). Data are shown as single data points for each oocyte and as means ± s.e.m.

Figure 5. Structural model of the K_V7.2 channel

K_V7.2 channels were generated based on the coordinates of K_V1.2 (PDB ID 2A79; Long et al. 2005). The transmembrane segments S1–S4 of one subunit are coloured (S1 (yellow), S2 (orange), S3 (red), S4 (pink) and the pore domain of the adjacent subunit (S5 (green), selectivity filter/pore helix (light blue), S6 (blue)). A, overview of 4 subunits. B and C, magnifications show the putative positions of the residues E119 and S122 in the outer S1 segment and their possible electrostatic interaction or formation of hydrogen bonds with R201 in S4 in top view and side view. Residues E119, S122 and R201 are shown in stick representation (CPK colour code) and putative electrostatic interactions or h-bonds are depicted as green dashed lines. The sequence of K_V7.2 is given underneath using the same colours as in the structural model for transmembrane segments. Residues E119, S122 and R201 are printed in bold and underlined. Residues in S1–S4 used to provide flexibility to the model are underlayed in grey.

Figure 6. Firing properties in a one compartment neuronal model cell

A and B, model cells are continuously stimulated by the current I_stim from time t = 0 ms. The M-current, I_M, is represented by K_V7.2/K_V7.3 (A) or E119G/K_V7.2/K_V7.3 (B) channels (parameters in Table 2). The duration of the evoked action potential burst is relatively prolonged in cells in which I_M is mediated by E119G/K_V7.2/K_V7.3 channels. I_stim= 1 μA cm⁻². C, burst duration as a function of I_stim. The increase in burst duration with increasing I_stim is steeper in cells that are simulated using the mutant channel. There is a threshold above which the M-current is insufficient to terminate the burst. This threshold is lower in simulations with parameters for mutant channels (continuous line K_V7.2/K_V7.3, dotted line E119G/K_V7.2/K_V7.3).