Gating Currents from K_v7 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_v7.2 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_v7.4 channels, highly related to K_v7.2 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 S₄ 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 S₄ 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.

Introduction

Translocation of charged particles (termed gating charges) within the membrane electric field is responsible for activation of the conductance in voltage-gated ion channels (VGICs). Positively charged lysines (K) or arginines (R) within the fourth transmembrane segment (S₄) are the main gating charges in voltage-gated Shaker K⁺ channels (1–4) and possibly in most Na⁺, Ca²⁺, and K⁺ channels (5,6). In addition to S₄ charged residues, negatively charged residues in S₂ (2) and hydrophobic residues in S₁, S₂, and S₃, defining the extent of the electric field where the charges translocate (7–10), have been shown to contribute to the gating process; thus, the region comprising S₁ through S₄ represents the voltage-sensing domain (VSD) of VGICs.

Changes in channel gating represent a common pathogenetic mechanism for genetically inherited channelopathies affecting heart rhythm, skeletal muscle contraction, or neuronal excitability. In particular, gating alterations play a major role in the pathogenesis of benign familial neonatal seizures (BFNS) (11–14), a rare autosomal-dominant idiopathic epilepsy of the newborn caused by mutations in the genes encoding for K_v7.2 (15,16) or, more rarely, K_v7.3 (17). K_v7.2 and K_v7.3 subunits underlie the M-current (I_KM), a slowly activating and deactivating K⁺ current that regulates neuronal excitability in the subthreshold range for action-potential generation. I_KM-forming K_v7.2 and K_v7.3 subunits are widely distributed in hippocampal, neocortical, and cerebellar neurons, at key sites for network oscillations and synchronization control (18); in these neurons, in addition to somatodendritic compartments, K_v7.2 and K_v7.3 subunits appear to be mainly localized at the axon initial segment (19), at Ranvier nodes (20), and at synaptic terminals (21). Among the other members of the K_v7 gene family, K_v7.1 is mainly expressed in the heart muscle and K_v7.4 in cochlear and vestibular organs of the inner ear, as well as in central auditory pathways, visceral and vascular smooth muscle, and skeletal muscle, whereas K_v7.5, in addition to neurons, has been also detected in human adult skeletal muscle and in vascular smooth muscle cells (22).

In K_v7.2, most BFNS-causing mutations are localized either in the large intracellular C-terminal domain, a critical region for subunit assembly and channel regulation, or in the VSD (14). A mild decrease of I_KM appears sufficient to cause BFNS, and haploinsufficiency seems the primary pathogenetic mechanism for both familial and sporadic forms of the disease. Although some mutations affect intracellular protein trafficking and polarized targeting (23,24), others reduce channel sensitivity to voltage and stabilize the closed-state conformation (11–13). However, genotype-phenotype correlations in BFNS-affected individuals are weak, and the intimate causes for the complex constellation of neurological signs and symptoms in K_v7.2-mutation carriers are only poorly understood.

Functional consequences of disease-causing mutations in K_v7 channels have been mostly studied by means of macroscopic current measurements, a technique unable to provide a detailed assessment of the subtle functional changes in voltage sensing and of the potential structural consequences underlying altered gating behavior; instead, analysis of the gating currents can provide a direct measure of gating-charge translocation during VSD displacement upon channel gating. In this work, we performed macroscopic and gating-current measurements in K_v7 channels carrying mutations responsible for BFNS and/or peripheral nerve hyperexcitability (PNH) (11,25–27). Since gating currents from K_v7.2 channels could not be resolved, the experiments were carried out in the functionally-and structurally-related K_v7.4 channels.

The data obtained show that mutations affecting charged residues located in the more distal portion of S₄ decrease the stability of the open state and of the active VSD configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S₄ caused gating-pore currents activating at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying mutations at different VSD locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases.

Materials and Methods

Isolation of Xenopus oocytes

The dissociation, maintenance, and microinjection of Xenopus oocytes followed standard procedures (28).

cDNA transcription and oocyte injection

K_v7 cDNAs were cloned in pTLN vectors. Mutations were introduced by PCR using degenerated primers (QuickChange, Stratagene, Milan, Italy) and verified by sequencing. Plasmid linearization and in vitro transcription followed standard procedures (28).

Vaseline-gap cut-open voltage-clamp electrophysiology

Ionic and gating currents from Xenopus oocytes were measured at 28°C, as previously described (29,30), using a Peltier device with a negative-feedback thermistor as a temperature sensor. The composition of the external and internal solutions for ionic current recordings were (in mM) 101 N-methy-D-glucamine (NMG), 12 KOH, 4 Ca(OH)₂, and 20 Hepes, pH 7.4 with methane sulfonic acid (MES acid), and 120 KOH, 2 EGTA, and 20 Hepes, pH 7.4 with MES acid, respectively. For gating-current recordings, the external and internal solutions contained (in mM) 100 tetraethylammonium-hydroxide (TEA-OH), 2 Ca(OH)₂, 2 Ba²⁺, and 20 Hepes, pH 7.4 with MES acid, and 115 TEA-OH, 2 EGTA, and 20 Hepes, pH 7.4 with MES acid, respectively. Oocytes were permeabilized by adding 0.3% saponin to the lower chamber for ∼1 min. Microelectrodes were pulled from borosilicate glass capillary tubes to obtain a resistance of 0.1–0.5 MΩ when filled with 3 M CsMES + 20 mM CsCl. Ionic currents were activated by depolarizing pulses of 1.5-s duration from −100 to +40 mV from a holding potential of −90 mV, followed by an isopotential pulse at −90 mV of 800–1500 ms. Capacity currents were compensated by analog circuitry and subtracted off-line. The data acquisition system was built in-house based on an Innovative Integration SB6711 with A4D4 boards running with in-house-written software. Ionic-current data were filtered at 1–2 kHz and sampled at 2–4 kHz. For gating-current recordings, oocytes were maintained at 0 mV for 30 min to deplete intracellular K⁺; after this period, the cells were exposed to the TEA-based solutions (± external Ba²⁺), and the following protocol was applied: from a holding potential of −90 mV, a 20-ms pulse to −100 mV was followed by voltage steps from −80 mV to +80 mV of 70-ms duration before a final 70-ms step to −100 mV was applied. Data were filtered at 5 kHz and sampled at 50 kHz. Capacity currents were compensated by analog circuitry and subtracted on-line using a p/−8 protocol from a subtracting holding potential of −100 mV.

Data analysis and statistics

To minimize the effect of extracellular K⁺ accumulation, conductance (G) values were calculated as previously described (28). G values were expressed as a function of membrane potential (G/V curves), and the obtained data were fit to a Boltzmann distribution of the following form: y = max/[1 + exp(zF(V_1/2 − V)/RT)], where V is the test potential, V_1/2 is the half-activation potential, z is the effective valence, and F, R, and T have their usual thermodynamic meanings. Activation and deactivation kinetics were analyzed by fitting the current traces with a single-or a double-exponential equation (12). For the traces fit with a double exponential (calculated with the equation τ = (τ_fA_f + τ_sA_s)/(A_f + A_s), a single time constant was thus obtained that represented the weighted average of the slow and fast components.

In gating-current recordings, the gating charge (Q) was calculated as the time integral of the gating currents (Ig) at each potential after leak subtraction. Q was plotted as a function of membrane potential (Q/V curves), and the data were then fit to the previously described form of the Boltzmann equation. Q_ON and Q_OFF kinetics were obtained by fitting the integrated Ig_ON and Ig_OFF traces, respectively, with a single-or a double-exponential equation.

Data are expressed as the mean ± SE of the given number of experiments (n). Data sets were compared using matched Student's t-tests or, if necessary, one-way ANOVA, followed by the Newman Keul's test. In the figures, values statistically different from those of the respective controls (p < 0.05) are indicated by asterisks (see Figs. 2 and 3).

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).

Computational modeling

Homology models of the VSDs of K_v7.4 and K_v7.4 R4Q channels were generated using the software package MODELER v9.8. Structural coordinates from K_v1.2 were used as a template. CHARMM-GUI provided scripts for building a membrane system with explicit solvent. The full system was energy minimized and then equilibrated for 0.23 ns. Upon equilibration, a free MD simulation was performed over 10 ns to observe the dynamics of our model.

Results

General features of the gating currents from K_v7.4 channels

K_v7 subunits display a core domain formed by six transmembrane segments (S₁–S₆), and cytoplasmic N- and C-termini (Fig. 1 A). S₄ primary sequence alignment among several K_v channels, including all K_v7 members, shows a variable number of positive charges (numbered 0–7) (Fig. 1 B). Characteristic in all subunits of the K_v7 subfamily is the presence of an uncharged Q residue at the position corresponding to R3 and a negatively charged D residue (D1) immediately before the R6 charge. Within the sequence shown, K_v7.2 and K_v7.4 subunits differ at only three positions, all occupied by hydrophobic residues; in the entire VSD, K_v7.2 and K_v7.4 primary sequences are ∼90% similar. This high degree of primary sequence homology between K_v7.2 and K_v7.4 must be highlighted as very relevant for the study of this sensor.

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.

In Xenopus oocytes at 28°C, both homomeric K_v7.2 and K_v7.4 channels give rise to voltage-dependent K⁺ currents that have a threshold of activation around −50 mV and display slow activation and deactivation kinetics (Fig. 1 C). After ionic current blockade with intracellular and extracellular perfusion using K⁺-free and tetraethylammonium (TEA)-based solutions in the presence of 2 mM extracellular Ba²⁺, fast and transient time- and voltage-dependent currents corresponding to the ON and OFF gating currents (Ig_ON and Ig_OFF, respectively) were recorded from K_v7.4-expressing cells (Fig. 1 D). Ig_ON showed a quasi-instantaneous activation followed by a decay that was faster during stronger depolarization; no residual current was detected at the end of a 70-ms depolarizing pulse to >0 mV. On the other hand, Ig_OFF showed a rising phase followed by a slower time-dependent decay. Notably, 2 mM Ba²⁺ did not influence Ig_ON and Ig_OFF properties of K_v7.4 channels; in fact, V_1/2 values were −26.7 ± 0.6 mV and −30.7 ± 0.9 mV, and z values were 1.93 ± 0.14 and 1.8 ± 0.14 for Q_ON/V with or without, respectively, extracellular Ba²⁺ (n = 4–10).

By contrast, no gating currents could be detected in oocytes expressing homomeric K_v7.2 channels (Fig. 1 D) or heteromeric K_v7.2/K_v7.3 channels (28), possibly because of a lower density of functional channels in the plasma membrane and a reduced temperature sensitivity of the activation kinetics of K_v7.2 channels when compared to K_v7.4 channels (28,31). Among K_v7 channels, K_v7.4 channels display the lowest single-channel conductance and opening probability (32); thus, the fact that the maximal density of macroscopic currents in K_v7.4- and K_v7.2/K_v7.3-expressing oocytes was similar suggests that the number of functional K_v7.4 channels was the highest among K_v7 channel subtypes. Moreover, it has been observed that most K_v7 channels appear to be present in the plasma membrane in a functionally silent (nonconductive) state, further complicating the interpretation of biochemical data (31,33).

Nonetheless, the fact that gating currents could not be recorded for K_v7.2, whereas these could be resolved adequately in K_v7.4-expressing oocytes, combined with the described sequence similarity in VSD primary sequence (particularly within S₄) between K_v7.2 and K_v7.4 subunits, prompted us to investigate to what extent K_v7.4 channel gating resembled that of K_v7.2 channels, and, in particular, to verify whether mutations in the VSD could prompt similar gating changes when incorporated into K_v7.2 or K_v7.4 channels. To this end, we neutralized the charge at the R2 position (R2Q mutation). In K_v7.2, such neutralization generated channels retaining all of the pore properties (single-channel conductance, opening probability, K⁺ selectivity, and sensitivity to blockade by external TEA) of wild-type channels, but it dramatically modified channel gating properties: K_v7.2 R2Q channels carried time-and voltage-independent currents (34) (Fig. 2 A), a result suggesting that the R2Q mutation locked the VSD in the active state. Gating changes identical to those described for K_v7.2 were also observed when the R2Q mutation was incorporated into K_v7.4 channels (Fig. 2 A). Similar functional consequences have been also observed when the corresponding mutation was introduced into K_v7.1 channels (35), allowing us to further generalize the fundamental role of the R2 charge in VSD resting-state stabilization in channels of the K_v7 subfamily. Moreover, although K_v7.4 and K_v7.4 R2Q channels exhibited similar macroscopic current amplitudes (Fig. 2 B), Ig_ON and Ig_OFF could not be recorded from K_v7.4 R2Q-expressing cells (Fig. 2 C), confirming that charge neutralization at R2 effectively impeded VSD return to the resting state.

Altogether, these results suggest the existence of marked analogies between homomeric K_v7.2 and K_v7.4 channels in the VSD structural transitions that occur during voltage sensing; therefore, K_v7.4 channels represent surrogates for K_v7.2 that are suitable for investigating the functional effects of disease-causing mutations on both ionic and gating currents.

Molecular mechanisms of channel dysfunction by K_v7.4 mutations in the C-terminal region of S₄

Based on the previous conclusions, we introduced at the corresponding positions of K_v7.4 some S₄ mutations that cause BFNS and/or PNH when present in K_v7.2. We initially focused on two mutations affecting residues located in the S₄ C-terminal region: R6W, a heterozygous germ-line mutation responsible for BFNS in two children from the same family (26), and D1G, found in a sporadic case of BFNS (27), which affected the negatively charged aspartate immediately preceding R6. Moreover, to investigate the differential role of charge and bulkiness at R6, the R6Q mutation was also studied.

At the macroscopic current level, all three mutations (R6W, R6Q, and D1G) caused rightward shifts of ∼30 mV in the V_1/2 of the G/V curves of Kv7.4 channels (Fig. 3, A and B, and Table 1). Activation and deactivation kinetics for R6W, R6Q, and D1G channels were significantly faster than for wild-type channels (Fig. 2, D and E, and Table 1). Similar functional changes were described when the corresponding mutations were introduced in K_v7.2 channels. In fact, the macroscopic currents carried by D1G- (27), R6Q- (34), and R6W-carrying K_v7.2 channels (36) displayed a reduced sensitivity to voltage in their steady-state G/V curves, together with an acceleration of both activation and deactivation kinetics.

Table 1.

Gating properties of S₄ K_v7.4 mutants

	G					QON				QOFF
	n	V1/2 (mV)	z (e−)	Activation τ (ms)	Deactivation τ (ms)	n	V1/2 (mV)	z (e−)	Q at +40 mV (nC)	V1/2 (mV)	z (e−)	Q at +40 mV(nC)
Wt	10	−18.4 ± 0.7	2.04 ± 0.11	212 ± 37∗ (0 mV)	130 ± 11 (−20 mV)	10	−24.1 ± 0.5	1.93 ± 0.14	1.2 ± 0.2	−28.4 ± 0.5	1.9 ± 0.13	1 ± 0.3
					21.7 ± 2 (−100 mV)
R4Q	4	+16 ± 1∗	1.43 ± 0.48∗	576 ± 31∗ (0 mV)	258 ± 31∗ (−20 mV)	10	nd	nd	nd	+12 ± 1∗	1.51 ± 0.16∗	0.62 ± 0.1∗
R4W	4	+32.3 ± 0.7∗	1.81 ± 0.48	6590† (0 mV)	3866† (−20 mV)	5	nd	nd	nd	nd	nd	0.26 ± 0.01∗
D1G	4	+9.1 ± 0.5∗	2.18 ± 0.07	37 ± 11∗ (0 mV)	27 ± 5∗ (−20 mV)	4	−17.9 ± 2.3∗	1.38 ± 0.22∗	0.8 ± 0.2	−13.2 ± 1.7∗	1.38 ± 0.12∗	1 ± 0.3
					6.8 ± 1.4∗ (−100 mV)
R6Q	6	+11.2 ± 0.7∗	2.01 ± 0.16	142 ± 12∗ (0 mV)	41 ± 6∗ (−20 mV)	7	−15.9 ± 0.9∗	1.44 ± 0.04∗	0.84 ± 0.2	−15.9 ± 0.5∗	1.39 ± 0.06∗	0.93 ± 0.2
					14.8 ± 3.6∗ (−100 mV)
R6W	6	+11.1 ± 1.4∗	1.47 ± 0.16∗	134 ± 27∗ (0 mV)	54 ± 12∗ (−20 mV)	5	−11 ± 1.5∗	1.03 ± 0.07∗	1.3 ± 0.3	−14.5 ± 1.1∗	1.09 ± 0.06∗	1.2.± 0.3
					10.9 ± 3.3∗ (−100 mV)

nd, not determined.

^∗Significantly different (p < 0.05) from the corresponding value in wild-type Kv7.4 channels.

^†Due to the extremely slow activation and deactivation kinetics for the R4W channels, the data shown are from a single cell in which longer (8-s) depolarizing and repolarizing pulses were utilized.

Gating-current analysis in K_v7.4 channels carrying the R6W, R6Q, or D1G mutations showed a right shift by 13, 8, and 6 mV, respectively, in the V_1/2 of the Q_ON/V relationships (Fig. 3, A and C, and Table 1). Qualitatively similar results were observed in the V_1/2 of the Q_OFF/V (Table 1). All three mutations reduced the slopes of the Q_ON/V and Q_OFF/V relationships, with the R6W causing the largest degree of change (Table 1). Ig_ON decay kinetics in R6Q and D1G channels were not affected; the time constants were 5.9 ± 0.5 ms, 5.4 ± 0.4 ms, and 5.7 ± 0.6 ms for wild-type, R6Q, and D1G channels, respectively. Only for R6W channels were Ig_ON decay kinetics slightly slower (10.7 ± 0.4 ms; p < 0.05 versus wild-type channels). As a result, the large difference between the slow macroscopic current activation kinetics and the fast Ig_ON decay, characteristic of wild-type channels, was reduced in all three mutants (Fig. 3 D). By contrast, Ig_OFF rising phase was largely abolished in R6W, R6Q, and D1G mutant K_v7.4 channels (Fig. 3 A); moreover, when compared to wild-type channels, Ig_OFF decay kinetics were also faster for all three mutant channels (Fig. 3 E). In all channels examined, Ig_OFF decay kinetics could be superimposed on those of ionic current deactivation (Fig. 3 E).

Molecular mechanisms of channel dysfunction by K_v7.4 mutations in the central region of S₄

In K_v7.2 channels, mutations affecting the R4 residue have been associated with hyperexcitability disorders. For example, the R4W mutation was observed in patients affected with BFNS and PNH (11), and the R4Q mutation was noted in a family affected by PNH in the reported absence of BFNS (25).

Ionic currents from R4Q or R4W Kv7.4 channels displayed a positive shift in their V_1/2, which was larger in R4W (∼50 mV) than in R4Q (∼30 mV) channels (Fig. 4, A and B, and Table 1). A decrease in activation and deactivation rates was also observed, which appeared more dramatic for R4W-than for R4Q-carrying channels; similar changes in the kinetics and steady-state properties of the macroscopic currents were observed when the corresponding mutations were incorporated in Kv7.2 channels (11,25). After ionic current suppression in both R4Q and R4W channels, depolarizing pulses to >0 mV revealed the appearance of nonlinear currents with complex kinetics; the fast transient Ig_ON was followed by a persistent noninactivating current whose magnitude increased upon further membrane depolarization (Fig. 4, C and D), thus preventing a detailed analysis of Ig_ON kinetics and Q_ON/V properties. At +80 mV, the magnitude of this current was four and two times larger in R4Q and R4W channels, respectively, when compared to wild-type channels. Upon membrane repolarization to −100 mV, in both mutant channels, an instantaneously rising Ig_OFF was recorded, which decayed with a double-exponential time constant, having a fast (∼0.5-ms) followed by a slower (∼12-ms) component (data not shown); the Q_OFF/V curve was right-shifted by ∼40 mV, also showing a slightly decreased steepness (Table 1).

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.

The magnitude of the depolarization-induced persistent outward current was directly proportional to the number of channels in the oocyte membrane (estimated by Q_OFF measurements) (Fig. 4 E), suggesting that the current flowed through these same channels. No apparent changes in the reversal potential of the persistent outward current from R4Q mutant channels were observed when Na⁺, K⁺, or Li⁺ were present in the extracellular solution, suggesting poor current selectivity for monovalent cations (data not shown); the fact that both internal and external solutions were devoid of Cl⁻, an experimental condition chosen to minimize the contribution of Ca²⁺-dependent and Ca²⁺-independent Cl⁻ currents abundantly expressed in the oocyte membrane, rules out a significant contribution of Cl^- ions.

Despite these technical limitations, the fact that the persistent noninactivating currents were larger than those recorded in oocytes expressing wild-type or any other mutant K_v7.4 channels, and that their magnitude was related to the number of functional channels, strongly suggests that they correspond to gating-pore currents similar to those generated upon the removal of specific basic residues in the VSD in several VGICs (37–42). The putative pore reported here opens on depolarization, indicating that the involved R4 residue occupies a deeper position in the resting state of the K_v7.4 VSD.

Finally, attempts to identify the depolarization-activated persistent currents caused by the R4Q mutation in the K_v7.2 channel background were not successful, likely because of the described low density of K_v7.2 channels in the oocyte membrane (data not shown).

Modeling and molecular dynamics of the K_v7.4 VSD

To follow up on the possibility of a gating pore, we generated a structural homology model for K_v7.4 using K_v1.2 as a template (43). Molecular dynamics (MD) simulations were performed on this homology model in an all-atom explicit-solvent environment to observe the region surrounding R4. In the activated configuration, the K_v7.4 VSD formed two hourglass-shaped water crevices separated by a plug (Fig. 5, A and B). A salt bridge between R4 and E136 in S₂ (Fig. 5, A and B) was also observed, which is responsible for the focused electric field in the membrane (37,44,45). Movie S1 shows that in K_v7.4 channels, the VSD is poorly permeant to water, allowing only very brief episodes of water flow during the entire 10-ns MD simulation. On the other hand, the R4Q mutation, by impeding the ionic interaction between R4 and E136, caused the upper and lower water crevices to nearly join as the plug dissolved (Fig. 5 C); MD experiments in K_v7.4 R4Q channels show a persistent water flux across the crevices throughout the 10-ns simulation (Movie S2).

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.

Discussion

Mutation-induced changes in gating of VGICs often provide plausible pathogenetic explanations for channelopathies. In particular, changes in the sensitivity to voltage of homomeric K_v7.2 or heteromeric K_v7.2/K_v7.3 channels have been suggested to play a relevant role in the pathogenesis of BFNS as well as PNH (11–14). Gating-current analysis offers an in-depth view of channel gating properties, since it provides a direct assessment of VSD displacement during channel gating; however, to our knowledge, this technique has never been applied to the functional study of BFNS- or PNH-causing mutations. In this study, BFNS- and/or PNH-causing K_v7.2 mutations were investigated using K_v7.4 channels to correlate the mutation-induced changes in macroscopic current behavior with the altered function of the VSD, since gating currents from K_v7.2 channels could not be resolved.

When compared to K_v7.2, K_v7.4 channels show a high degree of sequence similarity in the VSD (∼90%) and an identical distribution of charged residues within S₄; moreover, mutations in the VSD cause similar functional changes in gating in K_v7.2 or K_v7.4. In fact, charge neutralization of the R2 residue generated channels carrying voltage- and time-independent currents when incorporated in K_v7.2 (34) or K_v7.4 channels, as well as in K_v7.1 channels (35). This functional similarity revealed by the R2Q mutation appears unique among K_v7 channel members; indeed, in Shaker channels, the simultaneous neutralization of the R1, R2, and R3 charges is required to generate voltage-independent currents (46), a result suggestive of a lesser role of the R2 charge alone in resting-state VSD stabilization in Shaker channels; however, it should be remembered that the R3 residue in K_v7 channels is naturally neutralized by a Q residue.

Macroscopic currents from homomeric channels formed by all members of the K_v7 family are characterized by slow activation and deactivation kinetics, and by the absence of inactivation (22), except in the case of K_v7.1, which does exhibit inactivation (47). Gating current recordings from K_v7.4 channels revealed a poor coupling between charge movement and ionic currents, with Ig_ON decay being at least 10 times faster than ionic current activation. This indicates that the slow activation gating of K_v7.4 channels is not due to a slow movement of the VSD, and it suggests the existence of slower and less voltage-dependent transitions among closed states near the open state, a result also supported by the existence of a rising phase in Ig_OFF (28). Moreover, the fact that Ig_OFF kinetics closely match those of ionic current deactivation also suggests that the closure of the pore may be rate-limiting for the VSD return to the resting state. In addition, poor coupling between VSD displacement (fast) and pore opening (slow) might also be responsible for the differences in macroscopic current kinetics observed between K_v7.2 and K_v7.4 homomeric channels (see Fig. 1); whether differences in S₄ uncharged residues, which are known to influence macroscopic gating in K_v7 cannels (13), contribute to this differential macroscopic current kinetics is yet to be explored (48).

In this work, we show that three mutations affecting charged residues located in the more distal portion of S₄ (R6Q, R6W, and D1G) similarly increased the rate of current activation and deactivation and decreased the sensitivity to voltage of the ionic current steady-state activation process by ∼30 mV. Gating-current measurements from channels carrying these mutations revealed a slight (∼10-mV) shift toward more depolarizing potentials and a decreased slope of the Q_ON/V curve; only minor effects were prompted by these mutations on Ig_ON decay kinetics, whereas Ig_OFF rose instantaneously and decayed faster. These results suggest that these mutations decrease the stability of the open state and of the active VSD configuration; however, the fact that they only caused a relatively mild rightward shift in the Q/V compared to the G/V curves suggests that the D1 and R6 residues do not directly participate in voltage sensing but rather may be involved in stabilizing the activated configuration of the VSD and allowing its slow electromechanical coupling with the pore opening process (27,43). Homology modeling of the K_v7.4 subunit to the open configuration of K_v1.2 revealed that R6 and D1 form ionized hydrogen bonds that stabilize the activated state of the VSD; in particular, R6 interacts with E146 in S₂, whereas D1 interacts with G222, W224, and K225 in the S₄-S₅ linker. Neutralization of R6 or D1 by BFNS-causing mutations would impede the formation of these hydrogen bonds, thereby leading to a marked destabilization of the active VSD configuration (Fig. S1 in the Supporting Material). In K_v1.1 channels, the R6Q mutation generated nonfunctional channels because of a maturation deficit; the R6K mutant, instead, like the R6 mutants herein described, accelerated Ig_OFF kinetics (49). In the same channels, mutations affecting residues located in the S₄ (50), the C-terminal region of the S₄-S₅ linker (51,52), the S₅ (53,54), and the S₆ (52,55) domains involved in stabilizing the activated state of the VSD all remove the Ig_OFF slow rising phase.

On the other hand, mutations affecting the R4 residue (R4Q and R4W), located more proximally along the S4 primary sequence and belonging to the first four voltage-sensing gating charges (1,2), dramatically decreased the ionic current activation and deactivation rates of K_v7.4 channels, with effects similar to those occurring in K_v7.2 (11,25,34). Upon pore current blockade, both R4Q and R4W channels generated very fast Ig_ON, followed by a small sustained current when depolarized. Upon membrane repolarization, Ig_OFF rose instantaneously and decayed rapidly; compared to wild-type channels, the Q_OFF/V curve for R4Q channels was right-shifted by +40 mV, also displaying a decreased slope. The sustained currents were specifically carried by R4Q or R4W channels, since they were never observed in oocytes expressing any other channel and, more important, their magnitude was directly proportional to the number of functional channels. Currents that have similar properties have been described in several VGICs upon the removal of specific basic residues in the VSD (37–42). These currents, defined as gating-pore currents or ω currents, flow through water-filled regions of the protein (crevices or gating pores) created by specific conformations of the VSD and where the electric field is highly concentrated. In Shaker channels, although the substitution of R1 with a histidine residue generated a proton-selective pore that opened upon membrane hyperpolarization (37), the introduction at the corresponding position of smaller hydrophobic residues (A, C, S, or V) allowed permeation by alkali cations (Cs⁺, Na⁺, K⁺, and guanidinum) (38). By contrast, depolarization-induced proton-selective currents were observed in R4H mutant Shaker channels (56). In our case, neutralization of R4 was associated with gating-pore currents activating at depolarized potentials, and it seems likely that the absence of the positive charge at R3, naturally replaced by a Q in all K_v7s, helps in the formation of the gating pore. It has been recently shown that neutralization of two sequential positively charged S4 Rs support gating-pore currents in Shaker channels (57). In a similar way, brain Na_v1.2 channels carried gating-pore currents upon membrane hyperpolarization when both R1 and R2 were neutralized, and upon membrane depolarization when R2 and R3 neutralizing mutations were introduced in domain II (39).

MD experiments confirmed the formation of a water pore in the active K_v7.4 R4Q VSD configuration, mainly as a consequence of the lack of an electrostatic interaction between R4 and the E136 residue in S2 (58); the resulting proximity of water molecules dramatically enhances ionic conduction across the crevices.

The results presented here, showing that gating-pore currents can be generated by mutations affecting R4 in the VSD of K_v7 channels, in addition to their structural implications, may be of crucial pathophysiological importance. Among over 80 K_v7.2 BFNS mutations, only two, both occurring at the R4 residue, have been described in families affected by skeletal muscle myokymia after BFNS (R4W (11)), or in the reported absence of BFNS (R4Q (25)); therefore, it seems likely that the specific functional consequences prompted by these mutations, which in addition to the gating-pore currents herewith described also include a positive shift in the voltage-dependent activation of the current and a dominant-negative effect exerted by mutant subunits on the wild-type channels (11,25), might contribute to the occurrence of myokymia. In fact, although K_v7 channels exert most of their physiological function around threshold values of membrane potentials, VSD displacement during prolonged depolarizations would trigger the formation of a gating pore and allow inward currents carried mainly by Na⁺ ions. Such inward Na⁺ currents would provide a persistent depolarizing force that would contribute to the axonal hyperexcitability characterizing the myokymic phenotype. Similar effects would also be triggered if protons, rather than Na⁺ ions, significantly permeated the gating pore (59). K_v7.2 channels are expressed at nodes of Ranvier in peripheral myelinated fibers, particularly in large motor axons, and their activation reduces intrinsic excitability (60). Gating-pore currents in S₄ mutants of Na_v1.4 channels contribute to hypokalemic (40,61) or normokalemic (41) periodic paralysis in skeletal muscle (42). Moreover, persistent inward Na⁺ currents have been identified in Na_v1.1 channels carrying gain-of-function mutations responsible for generalized epilepsy with febrile seizure plus, another epileptic channelopathy (62).

In conclusion, the results presented here reveal what we believe are novel insights into the molecular mechanism of voltage sensing in K_v7 channels, provide plausible and previously unexplored biophysical mechanisms to account for the pathogeneses of hyperexcitability diseases caused by mutations in the VSD of these channels, and might be of relevance for the development of drugs targeting K_v7 VSD and acting as gating modifiers (36). With this in mind, gating-pore blockers have been recently proposed as therapeutically useful in preventing or relieving attacks of weakness in patients with hypokalemic periodic paralysis caused by mutations in the VSD of the skeletal muscle Na_v1.4 channels (63).