Metal Bridge in S4 Segment Supports Helix Transition in Shaker Channel

Abstract

Voltage-gated ion channels play important roles in physiological processes, especially in excitable cells, in which they shape the action potential. In S4-based voltage sensors voltage-gated channels, a common feature is shared; the transmembrane segment 4 (S4) contains positively charged residues intercalated by hydrophobic residues. Although several advances have been made in understating how S4 moves through a hydrophobic plug upon voltage changes, the possible helix transition from α- to 3₁₀-helix in S4 during the activation process is still unresolved. Here, we have mutated several hydrophobic residues from I360 to F370 in the S4 segment into histidine, in i, i + 3 and i, i + 6 or i, i + 4 and i, i + 7 pairs, to favor 3₁₀- or α-helical conformations, respectively. We have taken advantage of the ability of His to coordinate Zn²⁺ to promote metal ion bridges, and we have found that the histidine introduced at position 366 (L366H) can interact with the introduced histidine at position 370 (stabilizing that portion of the S4 segment in α-helical conformation). In the presence of 20 μM of Zn²⁺, the activation currents of L366H:F370H channels were slowed down by a factor of 3.5, and the voltage dependence is shifted by 10 mV toward depolarized potentials with no change on the deactivation time constant. Our data supports that by stabilizing a region of the S4 segment in α-helical conformation, a closed (resting or intermediate) state is stabilized rather than destabilizing the open (active) state. Taken together, our data indicates that S4 undergoes α-helical conformation to a short-lived different secondary structure transiently before reaching the active state in the activation process.

Significance

Conformational transitions between α-helix and 3₁₀-helix in the segment 4 (S4) of Shaker potassium channel during gating has been under debate. This study shows the coordination by Zn²⁺ of a pair of engineered histidine residues (L366H:F370H) in the intermediate region of S4 in Shaker, favoring α-helical conformation. In the presence of 20 μM of Zn²⁺, the activation currents of L366H:F370H channels become slower, with 10 mV positive shift in the voltage dependence and no effects on deactivation time constants suggesting a stabilization of a closed state rather than destabilization of an open (active) state. Collectively, our data indicate that S4 undergoes secondary structure changes, including a short-lived secondary structure transition when S4 moves from the resting to the active state during activation.

Introduction

Voltage-gated ion channels (VGIC) play several roles in physiological processes, especially in excitable cells, shaping the generation and propagation of action potentials (1). It has been shown by biochemical, electrophysiological, and structural biology experiments that VGIC are formed by four subunits (or domains), each one containing six transmembrane segments (S1–S6) (2, 3). In each subunit, segments 1–4 (S1–S4) constitute the so-called segment 4 (S4)-based voltage sensor domain (VSD), and the segments S5 and S6 form a fourth of the pore domain. In S4-based VSDs, S4 contains positively charged residues (mainly constituted by arginine side chains), every three residues between hydrophobic residues. These charged residues sense the electric field across the membrane and move upon depolarization, conferring to the pore the ability to conduct ions (4). Segment S2 contains a negative residue (E283) that forms salt bridges with Arg (R) residues from the S4 segment stabilizing and guiding their movement (5). Also, it has been demonstrated that S1, S2, and S3 segments contain a set of hydrophobic residues that form the “hydrophobic plug,” through which S4, the side chains of the R residues, must pass to go from the resting to the active state, the latest linked to the increase in the open probability of the pore domain (6, 7, 8, 9).

Recently, crystal structures of some VGICs have been solved, and it has been noted that the S4 segments from different VGICs show different secondary structures. For example, in Kv1.2, a mammalian voltage-gated potassium channel that is homologous to a potassium channel from fruit fly named Shaker, S4 appears in α-helical conformation in its N-terminal half, whereas its C-terminal half exhibits 3₁₀-helical conformation (7). In NavAb, a bacterial sodium channel from Arcobacter butzleri, the entire S4 segment is shown in 3₁₀-helical conformation (10). In Ci-VSP, a VSD that connects and controls an intracellular phosphatase in Ciona intestinalis, the S4 segment is shown in α-helical conformation in its entirety in two structures, one near the resting state and the other near the active state (11). The human sodium channels have four homologous domains I–IV, each domain containing six transmembrane segments similar to a potassium channel subunit. The structure of the human sodium channel Na_V1.4 has been reported, and it shows that the S4 segment is organized in 3₁₀-helical conformation between second and sixth Arg in S4 (R2 and R6) for all domains but the N-terminal region of the S4 segment in domain III and IV in α-helical conformation (around R1, first Arg in S4) (12).

From a functional data perspective, by using His scanning on S4 and analyzing periodicity of the perturbation using gating currents (13), and by using metal ion bridges for putative intersegment interactions between S3 and S4 (8), it has been suggested that S4 from Shaker can adopt a transient 3₁₀-helical conformation during the activation process. Nonetheless, a direct physical measurement of the interconversion from α- to 3₁₀-helical secondary structure has not yet been determined for S4. An attempt to probe 3₁₀ conversion from α structure in Ci-VSP showed that the conversion is not required in this voltage sensor (14). Recently, using nonsense suppression to investigate the hydrogen bonding of the main chain of Shaker S4 segment, it has been found that the active conformation of segment S4 was destabilized when the hydrogen bond in residue 369 was perturbed (15). These data suggest that destabilization of the helical conformation interferes with the movement of the S4 segment from resting to the active state. Even though those reports have suggested a conformational transition in different regions when the S4 segment reaches the active state (8, 13), no other direct evidence of a conformational transition has been provided, which keeps the matter under debate.

Metal ion bridges and disulfide bonds have been widely used to probe movement and distances between different residues in VGIC proteins (8, 16, 17, 18, 19, 20, 21, 22). This method has been especially used to probe interactions between residues in segment S4 and S5 in Shaker (17) and K_V1.2 (19) as well as between segments S3 and S4 in Shaker (8, 20). We engineered a pair of histidine residues within the S4 segment for coordination with Zn²⁺ to determine transitions from α- to 3₁₀-helical conformation or vice versa in this transmembrane segment. We placed His residue pairs in different positions to favor one or the other helical conformation. In two cases (i, i + 3 and i, i + 6, where i is a residue number), the His-His pair are expected to be aligned in 3₁₀-helical structures and would stabilize the helix in this conformation when coordinated by Zn²⁺. In the other two cases (i, i + 4 and i, i + 7), Zn²⁺ is expected to coordinate the His-His pair in α-helical structures, stabilizing it. We have found strong evidence that the histidine introduced at position 366 (L366H) coordinates with the introduced histidine at position 370, therefore favoring the α-helical conformation (i, i + 4) in the presence of μM Zn²⁺ concentrations. The kinetics of the activation of K⁺ currents in L366H:F370H mutant channels were slowed down by a factor of 3.5, the voltage dependence was rightward shifted (∼10 mV), and deactivation time constant was unchanged when 20 μM of Zn²⁺ was present in external solution. These results imply that the stabilization of a region of the S4 segment in α-helical conformation counteracts the transition of VSD to its active state. Taken together, our data support the evidence that the S4 segment undergoes a secondary structural transition from α-helical to a short-lived secondary structural conformation, different from α-helical, when transitioning from the resting state to the active state during the activation process.

Materials and Methods

Site-directed mutagenesis

We used Shaker zH4 K⁺ channel with fast inactivation removed by deleting residues from 6 to 46, Δ6–46, cloned into pBSTA vector (23). Mutations were performed using QuikChange (Stratagene, La Jolla, CA). All plasmids containing WT or mutant Shaker complementary DNA were sequenced and then linearized by restriction enzyme NotI. Complementary RNA was transcribed using in vitro transcription kits (T7 RNA expression kit; Ambion Invitrogen, Austin, TX). After 12–24 h from harvesting and deffoliculation, oocytes at stages V–VI were injected with complementary RNA (5–100 ng diluted in 50 nL of RNase-free water). Injected oocytes were incubated from 1 to 3 days at 12 or 18°C, depending on the injected construct, in standard oocytes solution that contains the following components in mM: 100 NaCl, 5 KCl, 2 CaCl₂, 0.1 EDTA and 10 HEPES—the pH was set to 7.4. Standard oocytes solution was supplemented with 50 μg/mL gentamycin to avoid contamination during incubation.

The 3₁₀- and α-helix are distinct in their backbone hydrogen bonding pattern (24). In 3₁₀-helices, the carbonyl group of a residue i binds to the nitrogen from amide group in residue i + 3. In α-helices, the residue i from carbonyl group binds to the nitrogen from amide group in residue i + 4. Common features of canonical 3₁₀-helices are as follows: three residues per turn, the angle between the residues is 120° around the helical axis, and each turn is spaced by 6 Å. For α-helices, the common features are as follows: 3.6 residues per turn, the angle between the residues is 100° around the helical axis, and each turn is spaced by 5.4 Å. Thus, side chains of coupled residues spaced in i, i + 4 and i, i + 7 patterns tend to stabilize the α-helical conformation because they are closer to multiples of 3.6, and the angle between i and i + 4 is 400° and between i and i + 7 is 700°. On the other hand, side chains spaced in i, i + 3 and i, i + 6 patterns tend to stabilize the 3₁₀-helical conformation because the angle between residues i and i + 3 is 360° and between i and i + 6 is 720°. Considering His:His mutants arranged in i, i + 4 and i, i + 7, the side chains would be in opposite directions if S4 were in a 3₁₀-helical conformation, being an impediment for a side chain interaction. In contrast, His:His mutants arranged in i, i + 3 and i, i + 6, the side chains would also be in opposite directions if S4 were in an α-helical conformation. Therefore, the strategy of His:His mutants was intended to favor one or the other helical conformation depending on the position in which they are placed (25, 26, 27, 28, 29).

Electrophysiological recordings

Ionic currents were recorded using cut-open oocyte voltage-clamp method with a voltage-measuring pipette with resistance from 0.2 to 0.8 MΩ (30). Pipettes were pulled using a horizontal puller (P-87 Model; Sutter Instruments, Novato, CA). Capacitive transient currents were compensated by a dedicated circuit. Raw currents were analogically filtered at 20–50 kHz with a low pass four-pole Bessel filter built in the amplifier (Dagan CA-1B; Dagan, Minneapolis, MN). Processed currents were then sampled at 1 MHz using a 16-bit analog-to-digital converter (USB-1604; Measurement Computing, Norton, MA), digitally filtered with Nyquist frequency and decimated for an acquisition rate of 100–200 kHz. An in-house software was used to acquire (GPatch64MC) and analyze the data (Analysis). For ionic measurements, the external solution was composed by (mM): K-MES—methanesulfonic acid (MES) 12, Ca-MES 2, HEPES 10, EDTA 0.1, and N-methyl-D-glucamine (NMDG)-MES 108 (pH 7.4); and the internal solution was composed by (mM): K-MES 120, EGTA 2, and HEPES 10 (pH 7.4). To ensure the channels were closed before the depolarizing pulses, a conditioning hyperpolarized prepulse was used with different voltages (depending on the mutant), and the holding potential used was −80 or −100 mV, depending on the mutants. All recordings were performed at room temperature. Before use, Zn²⁺ was diluted to its final concentration in external solution in the absence of EDTA, from a 30 mM stock solution (ZnCl₂).

Data analysis

Macroscopic K⁺ currents were recorded from 4 to 11 oocytes per mutant, and their peak was transformed into conductance (G) by using the equation below:

$$G=\frac{I_m}{V_m-\frac{RT}{F}\ln \left(\frac{{\left[K^{+}\right]}_{out}}{{\left[K^{+}\right]}_{in}}\right)},$$ (1)

where I_m is the K⁺ current activated by membrane voltage V_m, R is the gas constant, T is the temperature in Kelvin, F is the Faraday constant, and [K]_in and [K]_out are the intracellular and extracellular K⁺ concentrations, respectively.

The K⁺ conductances were normalized, averaged, and plotted against V_m to build conductance-voltage (G-V) curves that were fitted with a two-state model by the equation below:

$$G\left(V_m\right)=\frac{1}{1+\exp \left(\frac{z_{app}F}{RT}\left(V_{1/2}-V_m\right)\right)},$$ (2)

where z_app is the apparent charge of the transition expressed in units of elementary charge (e₀), and V_1/2 is the voltage for 50% of the maximal conductance.

K⁺ currents during activation by depolarizing voltage pulses and during deactivation by hyperpolarizing voltage pulses were fitted by a double exponential shown below:

$$y\left(t\right)=y_0-A_1\exp \left(-\frac{t}{{\tau }_1}\right)-A_2\exp \left(-\frac{t}{{\tau }_2}\right).,$$ (3)

where A₁ and A₂ are the amplitudes from the first and the second exponential, respectively. τ₁ and τ₂ are the time constants for the first and second exponentials, respectively; γ₀ is a baseline adjustment.

For analysis purposes, we considered a weighted time constant, calculated by the following equation:

$${\tau }_w=\frac{A_1{\tau }_1+A_2{\tau }_2}{A_1+A_2},$$ (4)

where τ_w is the weighted time constant.

MATLAB (The MathWorks, Natick, MA) and Origin9.0 (Origin Lab, Northampton, MA) were used for calculating G-Vs, τ-Vs, plotting, and fitting the data. Data are shown as mean ± SEM.

Results

We tested the hypothesis whether the S4 segment of Shaker undergoes secondary conformational transitions during the activation/deactivation processes. To that end, we replaced pairs of noncharged residues located at different positions in S4 helix by pairs of His (Fig. 1, A and B). Each pair of residues replaced by His were chosen so that the His:His coordination by Zn²⁺ would be favored exclusively in α-helical or 3₁₀-helical conformations of the secondary structure of the S4 region (Fig. 1 A). For example, when a His:His pair was placed every four or seven residues apart (i, i + 4 or i, i + 7), a possible interaction between them would favor an α-helical conformation. On the other hand, when they were placed every three or six residues apart (i, i + 3 or i, i + 6), they would favor a 3₁₀-helical conformation (see Materials and Methods). His:His were introduced in the fast inactivation removed version of the Shaker channel (Δ6-46, Shaker) (23). We analyzed possible His:His interactions using the voltage dependence of the K⁺ conductance (G-V curves) as well as the kinetics of activation and deactivation of the voltage-dependent K⁺ currents. If a conformation change were to occur during activation or deactivation, the possible stabilization of either secondary structure conformation by a His:His interaction may oppose or promote that process, possibly altering the activation/deactivation kinetics and/or voltage dependence. To enhance or disrupt His:His interaction, we exposed the mutant channels to different pHs. Acidic environment at a pH of 5.5 would protonate the majority of the exposed His residues, whereas basic environment at a pH of 9.2 would deprotonate the majority of the exposed His residues. Zn²⁺ was also used to coordinate the His:His by metal bridges (17, 27, 29).

Figure 1.

His:His screening in S4 segment. (A) His:His residues were mutated in S4. The top row in black shows the WT amino acid Shaker S4 sequence. Arg residues are shown in red. Colored rows show the residues mutated, and the dashes mean WT residues. Rows in purple represent the mutants intended to stabilize S4 in α-helical conformation, whereas rows in green represent the mutants intended to stabilize S4 in 3₁₀-helical conformation. Green and purple top bars represent the portion of S4 segment that are organized in α-helical or 3₁₀-helical configuration according to K_V1.2 VSD from a crystallography-based model (Protein Data Bank [PDB]: 3LUT), respectively. (B) Side view from S1 to S4 segments at the active/relaxed state in the K_V1.2 VSD from a crystallography-based model (PDB: 3LUT). For clarity purposes, helix backbones from S1 to S3 are shown in gray and from S4 in black. Residues mutated I360H (orange), V363H (green), L366H (red), V367H (magenta), and F370 (blue) are depicted with spheres.

Zn²⁺ affects K⁺ currents of L366H:F370H channel: possible stabilization of α-helical conformation

Remarkably, the only double mutant that showed evidence of His:His interactions, as measured by changes in voltage dependence and kinetics, was L366H:F370H in the presence of 20 μM Zn²⁺. Fig. 2, A and B show representative ionic current traces from L366H:F370H mutant in the presence (Fig. 2 B) and absence of 20 μM Zn²⁺ (Fig. 2 A). The Zn²⁺ effect on L366H:F370H was partially recovered after washout with external solution (containing 100 μM of EDTA to chelate free Zn²⁺) (Fig. 2 C). In the presence of Zn²⁺, the midpoint of L366H:F370H G-V curve is shifted ∼10 mV to more positive potentials (from −11.7 ± 1.3 to −0.3 ± 1.5 mV) without changing the slope of the curve (from 1.4 ± 0.1 to 1.2 ± 0.1), and the maximal conductance is decreased to ∼60% of the conductance of No Zn²⁺ condition (Fig. 2 D; Table 1). After extensive washing, the maximal conductance was recovered to 84% of the initial value (no Zn²⁺ condition). This is clearly different from the results of Zn²⁺ on wild-type (WT) or the single mutants L366H and F370H, in which no effects were observed (Fig. 2, E–G). These findings suggest that the coordination between L366H and F370H by Zn²⁺ is enough to oppose the activation process, indicating that a change in secondary structure conformation might be needed for that process.

Figure 2.

His:His coordination by Zn²⁺ between L366H and F370H in S4 segment. (A–C) Representative ionic currents for mutant L366H:F370H in external solution only (A), in the presence of 20 μM Zn²⁺ (B), and washout (external solution) (C). Inset in (C) is the voltage pulse protocol used to elicit the activation currents. (D–G) G-V curves for L366H:F370H (D), for WT (E), for L366H (F), and for F370H (G), in the absence of Zn²⁺ (No Zn²⁺, external solution—black squares), in the presence of 20 μM Zn²⁺ (Zn²⁺, red circles), and washout condition (Wash, external solution—blue triangles). G-V curves were normalized by the maximal G in the absence of Zn²⁺. Data are shown as mean ± SEM (N = 4–11). Continuous lines over G-V curves are the best fittings of Eq. 2 (see Table 1 for fitted parameters). To see this figure in color, go online.

Table 1.

G-V Fitted Values for All the Channels at Different Conditions

Channel	pH 7.4 (No Zn2+)						pH 5.5						pH 9.2						20 μM Zn2+
	V1/2 (mV)			z			V1/2 (mV)			z			V1/2 (mV)			z			V1/2 (mV)			z
WT	−19.1	±	0.7	2.3	±	0.1	4.8	±	0.9	2.3	±	0.2	−22.9	±	1.1	2.1	±	0.2	−14.2	±	1.1	2.1	±	0.2
I360H	−4.4	±	0.5	2.0	±	0.1	19.3	±	0.8	1.8	±	0.1	−8.0	±	0.7	1.8	±	0.1	−1.9	±	0.5	2.0	±	0.1
V363H	−0.4	±	0.9	1.0	±	0.0	29.8	±	1.2	1.0	±	0.0	−7.6	±	1.3	0.9	±	0.0	−0.4	±	0.9	1.0	±	0.0
L366H	−54.4	±	1.4	1.5	±	0.1	−24.3	±	0.6	1.6	±	0.1	−63.8	±	1.1	1.5	±	0.1	−55.2	±	1.4	1.4	±	0.1
V367H	0.0	±	0.6	3.0	±	0.2	30.4	±	0.6	2.8	±	0.2	−8.7	±	0.6	2.9	±	0.2	2.2	±	0.6	2.8	±	0.2
F370H	40.5	±	0.4	3.4	±	0.2	62.2	±	1.0	3.2	±	0.2	35.7	±	0.6	3.7	±	0.2	44.8	±	1.1	4.1	±	0.3
I360H:L366H	−32.1	±	1.2	1.9	±	0.1	−12.8	±	1.1	1.6	±	0.1	−39.5	±	1.2	2.0	±	0.2	−26.8	±	1.5	1.7	±	0.1
I360H:V367H	−2.3	±	0.7	3.6	±	0.3	28.7	±	0.5	3.4	±	0.2	−12.6	±	1.2	3.1	±	0.4	−1.4	±	0.7	3.4	±	0.3
V363H:L366H	−48.2	±	1.0	1.1	±	0.0	−19.7	±	1.1	0.9	±	0.0	−57.8	±	1.1	1.1	±	0.0	−42.0	±	1.2	0.9	±	0.0
V363H:V367H	−4.2	±	0.8	1.7	±	0.1	18.6	±	0.7	1.5	±	0.1	−12.7	±	1.0	1.7	±	0.1	−1.3	±	1.0	1.6	±	0.1
L366H:F370H	−11.7	±	1.3	1.4	±	0.1	18.3	±	0.8	1.5	±	0.1	−13.7	±	1.5	1.3	±	0.1	−0.3	±	1.5	1.2	±	0.1
V367H:F370H	38.9	±	0.5	4.6	±	0.4	56.2	±	0.2	4.2	±	0.1	35.3	±	0.5	4.7	±	0.4	40.5	±	0.4	4.1	±	0.2

All other double mutants were not clearly affected by either the presence of Zn²⁺ or by changes in the pH compared to the effect of these changes in WT channels (Figs. S1 and S2). The observed effects induced either by the presence of Zn²⁺ or pH changes were seen as modifications in the voltage dependence of G-V curves or in the activation time constants of K⁺ currents (Figs. S1 and S2). Even though the maximal conductance of L366H:F370H channels is affected by a pH of 5.5 (decreased to 60% Fig. S1 K), we could not infer whether the His:His were interacting in L366H:F370H channels at a pH of 5.5 because of the following: 1) the rightward shift on the G-Vs induced by a pH of 5.5 on WT, L366H, F370H, and L366H:F370H channels were very similar (∼25–30 mV Fig. S1, A, D, F, and K; Table 1); and 2) the activation time constants for WT, L366H, and L366H:F370H were similarly slowed down by the same factor, 1.3, but in the single mutant F370H, the time constants were slowed down by a factor of 4 (Fig. S2, A, D, F, and K). These results indicate that the effects of a pH of 5.5 on L366H:F370H cannot be clearly distinguished from the effects on L366H, F370H, and WT, and for this reason, we did not further pursue the pH effects.

Zn²⁺ metal bridge in L366H:F370H suggests a stabilization of closed states

To gain more insight about L366H:F370H interaction in the presence of Zn²⁺, the activation time constants were estimated by fitting a double exponential function (Eq. 3) to the rising phase of the ionic currents elicited by depolarizing membrane voltage steps (Fig. 2, A–C). Next, those time constants were weighted (Eq. 4) and plotted against membrane voltage (Fig. 3). Interestingly, in the presence of 20 μM of Zn²⁺, L366H:F370H activation currents become ∼3.5 slower than the currents (at +100 mV) in the absence of Zn²⁺ (Fig. 3 A). The activation time constants of WT and on the single mutants L366H and F370H channels in the presence of 20 μM of Zn²⁺ are on average only ∼1.3 slower than time constants in the absence of Zn²⁺ (Fig. 3, B–D). Taken together, the hypothetical stabilization by Zn²⁺ of an α-helical conformation between residues L366H and F370H may suggest that the transition from resting to active states in the S4 segment of the channel has been delayed, presumably stabilizing the closed (resting or intermediate) state.

Figure 3.

Zn²⁺ slows down activation time constants for L366H:F370H channel. Weighted activation τ-V curves in the absence of Zn²⁺ (No Zn²⁺, external solution—black squares), in the presence of 20 μM Zn²⁺ (Zn²⁺, red circles), and washout condition (Wash, external solution—blue triangles). (A) τ-V curves for L366H:F370H (B), τ-V curves for WT (C), τ-V curves for L366H (D), and τ-V curves for F370H. Time constants were calculated using Eq. 3 and posteriorly weighted using Eq. 4. Data are shown as mean ± SEM (N = 4–11). To see this figure in color, go online.

Because the activation time constants were fitted by the sum of two exponentials (Eq. 3), the analysis resulted in a fast and a slow component. We further investigate the effects of Zn²⁺ in each component. Fig. 4 A shows two normalized representative ionic current traces for L366H:F370H mutant at 100 mV in the absence (black trace) and in the presence of Zn²⁺ 20 μM (red trace). Zn²⁺ affects mainly the slow component, making it even slower (Fig. 4 B) and slightly increasing its proportion with respect to the fast component (Fig. 4 C). In addition, the slow component becomes fairly voltage independent in the presence of Zn²⁺, suggesting a limiting step introduced by the putative His:His coordination.

Figure 4.

Slow component of L366H:F370H ionic currents is more affected by Zn²⁺. (A) Normalized representative ionic currents from an oocyte for mutant L366H:F370H at 100 mV in external solution (No Zn²⁺—black line) and in the presence of 20 μM Zn²⁺ (red line). (B) Fast (open black square—No Zn²⁺, and open red circle—20 μM Zn²⁺—Zn²⁺ condition) and slow (filled black square—No Zn²⁺ and filled red circle—20 μM Zn²⁺—Zn²⁺ condition) component of activation time constant curves are shown. (C) Fraction of the slow component in the activation time constant in the absence (filled black square) and in the presence of 20 μM Zn²⁺ (filled red circles). Data are shown as mean ± SEM (N = 4-11). To see this figure in color, go online.

After 1 s at positive membrane voltage, the currents of L366H:F370H in the presence of Zn²⁺ have not reached a steady state and are decreased as compared to L366H:F370H currents in the absence of Zn²⁺ (Fig. 2, A–D). Therefore, we decided to extend the depolarizing voltage pulse to 5 s and elicit L366H:F370H currents at different Zn²⁺ concentrations to titrate its effect. Fig. 5, A–D show representative L366H:F370 current traces in the absence and in the presence of 5, 20, and 50 μM Zn²⁺, respectively. The higher the Zn²⁺ concentration in the bath, the larger is the effect on the currents (the current time courses become slower and the amplitudes smaller). The G-V is more rightward shifted (Fig. 5, E and F), and the proportion of the second component as well as the activation time constant are increased as the concentration of Zn²⁺ is increased in the bath (Fig. 5, G and H). These results indicate that the range of Zn²⁺ concentrations we have used (5–50 μM) is not enough to saturate its binding sites in the channels (His:His), an important information for modeling this effect.

Figure 5.

Concentration-dependent effects of Zn²⁺ on L366H:F370H ionic currents. (A–D) Representative ionic currents for mutant L366H:F370H in external solution (A), in the presence of 5 μM Zn²⁺ (B), in the presence of 20 μM Zn²⁺ (C), and in the presence of 50 μM Zn²⁺ (D). (E) G-V curves for L366H:F370H in the absence of Zn²⁺ (No Zn²⁺—black squares) and in the presence of 5 μM Zn²⁺ (red circles), 20 μM Zn²⁺ (blue triangles), and 50 μM Zn²⁺ (green diamonds). G-V curves were normalized by the maximal conductance (G) of the channels in the absence of Zn²⁺. Continuous lines over G-V curves are the best fittings of Eq. 2. The fitted parameters are as follows: V_1/2 = −11.7 ± 1.3 mV and z = 1.4 ± 0.1 in the absence of Zn²⁺; V_1/2 = −2.2 ± 1.3 mV and z = 1.3 ± 0.1 for 5 μM Zn²⁺; V_1/2 = 8.7 ± 1.2 mV and z = 1.2 ± 0.1 for 20 μM Zn²⁺; and V_1/2 = 22.8 ± 1.6 mV and z = 1.1 ± 0.1 for 50 μM Zn²⁺. (F) ΔV_1/2 was plotted against concentration. ΔV_1/2 was calculated using the following equation: ΔV_1/2 = ΔV_1/2ˍZn − ΔV_{1/2ˍNo Zn}. (G) Fraction of the slow component in the activation time constant in L366H:F370H in the absence of Zn²⁺ (No Zn²⁺—black squares) and in the presence of 5 μM Zn²⁺ (red circles), 20 μM Zn²⁺ (blue triangles), and 50 μM Zn²⁺ (green diamonds). (H) Fast (open symbols) and slow (filled symbols) component of activation time constant for L366H:F370H in the absence of Zn²⁺ (No Zn²⁺—black squares) and in the presence of 5 μM Zn²⁺ (red circles), 20 μM Zn²⁺ (blue triangles), and 50 μM Zn²⁺ (green diamonds). Data are shown as mean ± SEM (N = 3). To see this figure in color, go online.

Metal bridges can stabilize the closed state or the open state of the channel, depending on the position of the residues in the channel (17). Because Zn²⁺ promotes a rightward shift in the G-V curve and slows down the activation time constant for L366H:F370H, it can be speculated that in the presence of Zn²⁺, the resting state is stabilized. We measured the deactivation time constants of ionic currents for L366H:F370H mutant in the presence (Fig. 6 B) and the absence of Zn²⁺ (Fig. 6 A). The inset in Fig. 6 B is the voltage pulse protocol used to record K⁺ currents during deactivation by voltage. Two normalized representative ionic currents deactivating at different voltages −70 mV (left panel) and −150 mV (right panel) in the presence (red) and the absence (black traces) of Zn²⁺ are shown in Fig. 6 C. Interestingly, the time course of those currents practically superimposes, showing that deactivation is not affected by Zn²⁺. These results show that the backward rate, from open (active) to closed (resting or intermediate) state, is not affected by Zn²⁺, indicating a stabilization of a closed state by Zn²⁺. It is worth mentioning that currents from L366H:F370H mutant channels, in the absence of Zn²⁺ and the presence of EDTA (to ensure no coordination by heavy metals contamination of the experimental solutions) have a slower activation time constant and slower deactivation, and its voltage dependence (in steady state) is rightward shifted compared to WT channels (Figs. 3, A and B and 6 D).

Figure 6.

Deactivation time constants of L366H:F370H are not affected by Zn²⁺. (A and B) Representative deactivation current traces from an oocyte expressing the mutant L366H:F370H in the absence (A) and in the presence of 20 μM Zn²⁺ (B). Inset in (B) is the voltage pulse protocol used to elicit the currents. Dashed rectangle is the time window taken to show the representative current traces in (A and B). (C) Two normalized representative ionic currents at different voltages −70 mV (left panel) and −150 mV (right panel) in the presence (red line) and absence (black line) of Zn²⁺. (D) Deactivation time constant curves for L366H:F370H channel in the absence of Zn²⁺ (No Zn²⁺, external solution—filled black squares) and in the presence of 20 μM Zn²⁺ (Zn²⁺, open red circles). WT time constant in No Zn²⁺ condition is shown as open black circles. Time constants were calculated using Eqs. 3 and 4. Data are shown as mean ± SEM (N = 7). To see this figure in color, go online.

Discussion

Our functional data provide evidence that Zn²⁺ coordinates two engineered histidine residues, L366H and F370H, in the S4 segment in Shaker channels; this coordination is expected to stabilize that particular region of S4 segment in α-helical conformation. The crystallographic-based model of the structure of Kv1.2, a close homolog of Shaker potassium channel, shows that the S4 segments are in α-helical conformation in the N-terminal half and 3₁₀-helical conformation in the C-terminal half (7). The transition from α-helical to 3₁₀-helical occurs between residues V369 and F370 (7). Because the crystal structure of Kv1.2 (homologous of Shaker structure) was obtained at zero mV, the conformation is expected to be in the active/relaxed state. Based on functional data, it has been proposed that S4 undergoes conformational changes during the activation process (6, 8, 13). Recently, in a quest to show possible transitions between α- and 3₁₀-helical conformations in the S4 using Ci-VSP during activation, Kubota et al. (14) used Förster resonance energy transfer and luminescence resonance energy transfer to measure distances between the two ends of the S4 segments while it transits during the gating process. Those experiments showed no sign of change in the fluorescence signal that could support any conformational change. In addition, it has been suggested that in Shaker, the hydrophobic plug (a 10 Å thick hydrophobic region contributed by side chains of segments S1–S3) separates intra- from extracellular sides of the VSD (7, 9) and stabilizes the S4 region in 3₁₀-helical conformation as S4 moves through it during activation (8). Moreover, Infield et al. (15) have used nonsense suppression to investigate the main chain hydrogen backbone bonding within S4, and they have found that when they perturbed the hydrogen bond at position 369, the active conformation of segment S4 was destabilized. Nevertheless, this study did not provide direct evidence of conformational transitions in the S4 segment during its activation process.

The presence of 20 μM of Zn²⁺ did not significantly alter the voltage dependence and the activation time constants of K⁺ currents from WT channels nor from single mutants L366H or F370H channels (Figs. 2 and 3). We interpret these data, together with the clear effect of Zn²⁺ on L366H:F370H mutants, as strong evidence that there is indeed a coordination between L366H and F370H in the same channel by Zn²⁺. Our study used ionic currents activation that reflects the last concerted step of the VSD-to-PD electromechanical coupling mechanism. To gain more insight about the conformational changes of the L366H:F370H channel, the study of gating currents (directly related to VSD movement) in this channel would be ideal. However, because of their low expression in oocytes, we were unsuccessful in recording gating currents from this mutant.

If we assume that one His is protonated, it can serve as an organic cation and forms a cation-π interaction with deprotonated His⁰, whereas if His is deprotonated, the conjugative π-plane from imidazole side chain can interact with other deprotonated His by π-π stacking interaction. There is still a third possibility that both His are protonated, and their interaction is repulsive. Histidine imidazole group in aqueous solution shows a pKa around 6.5. It is unknown whether the His residues are buried into the membrane or exposed to aqueous crevices; therefore, pKa of His imidazole group in that region of the VSD is also unknown. Therefore, during our regular experiments at a pH of 7.4, it is possible that the side chain is either protonated (His⁺) or deprotonated (His⁰). For the pairs that did not show evidence of either Zn²⁺ or pH modulation (I360H:L366H, I360H:V367H, V363H:L366H, V363H:V367H, and V367H:F370H see Figs. S1 and S2), we cannot infer relative to 3₁₀-helix stabilization during VSD motion. Possible explanations for this lack of interaction include the following: 1) no conformational changes in these regions of S4 are required for activation, and 2) the geometry (orientation of side chains) is not appropriate for His to be coordinated by Zn²⁺. It should be added that the pH effects on the G-V curves of the channels studied here are consistent with the surface screening charge effects (31, 32, 33). To study possible interactions in an extended region of the S4 segment, we have also tried to record gating and ionic currents from the following His:His pairs: V363H:V369H, V363H:F370H, and I364H:F370H. However, none of these constructs has shown enough expression in Xenopus oocytes.

It has been suggested that using Ala scanning mutagenesis on C-terminal of S4 segment of drk1 voltage-gated K⁺ channel presents periodicity consistent with α-helical configuration (34). Moreover, it has been proposed that segment S4 has an intermediate state during activation and that S4 requires a short-lived secondary structural change to reach the active state (8, 13). Furthermore, by using His scanning mutagenesis, it has been suggested that the S4 segment of Shaker exhibits periodicity compatible with 3₁₀-helical configuration and that a transition to 3₁₀-helical configuration may be necessary to reach the active state (13). Our data support the idea that by stabilizing the α-helical conformation in the region of the third gating charge (R368) of the S4 segment has prevented a normally occurring secondary conformation (3₁₀-helix) change during the activation process based on our functional data using the His:His coordinated by Zn²⁺. The stabilization of S4 in α-helical in the presence of Zn²⁺ was enough to dramatically affect the kinetics of activation of L366H:F370H currents (Figs. 3 and 4), without changing deactivation time constant (Fig. 6), and promoted 10 mV rightward shift in voltage dependence (Fig. 2; Table 1). This also implies that closed states (resting or intermediate) were stabilized. Because the deactivation time constant was not affected by Zn²⁺, we infer that Zn²⁺ might not be able to coordinate with the His residues in the active state.

We have studied nine His:His mutants in the presence of Zn²⁺, six of them were successfully expressed in Xenopus oocytes (I360H:L366H, I360H:V367H, V363H:L366H, V363H:V367H, V367H:F370H, and L366H:F370H) and three were not (V363H:V369H, V363H:F370H, and I364H:F370H). Zn²⁺ has produced significant effects on only one (L366H:F370H) of the nine mutants studied, which is the basis of our claim of stabilization of the secondary structure of that S4 region in α-helix. In the resting (down) state, the outermost part of segment S4 in Shaker is surrounded by several residues (Asp, Cys, and Glu) that could potentially be part of the Zn²⁺ coordination (29) when our engineered His residue is present. Among these potential candidates, there are two Cys residues (C245 in S1, C286 in S2) and several Glu and Asp residues (E247 in S1, E283 in S2, E333, E334, E335, and D336 in S3–S4 extracellular loop, and E418 and E422 in S5). Shaker channels have a biding site for Zn²⁺, and this site receives no contribution of the native Cys residues C245 or C286 (35). Instead, the Zn²⁺ coordination site in Shaker includes the residues E247 and R365 and concentrations of Zn²⁺ higher than 0.1 mM (21). Even though the position R365 is one residue apart from L366, the proximity does not necessarily mean that L366H is coordinating with E247 because 1) the lack of effects attributable to Zn²⁺ on the single mutant L366H and 2) the residues R365 and L366H are oriented 100° apart in an α-helix or 120° in 3₁₀ configuration. Also, the effect we attribute to the coordination of Zn²⁺, in double mutant L366H:F370H, was present at a five times lower concentration of this divalent cation, 20 μM. The series of acidic residues E333, E334, E335, and D336 (EEED) are located in the S3–S4 extracellular loop, therefore far above the L366H:F370H region, where the Zn²⁺ coordination was observed. Using Cys mutation, Henrion et al. (8) have reported metal bridges in the open state between residues R365C and T326C. The residue T326, in S3, is located two turns below E333, the innermost Glu from EEED (7), suggesting R365C in the up state does not reach EEED, excluding, therefore, the possibility of interaction between 366H and those four acidic residues, especially in the resting and/or intermediate states. In addition, those positions (EEED) have been reported to be in close proximity to the S3–S4 linker, close to S4, because they quench a fluorophore placed at residue M356 (36), which is three turns above the region where the metal coordination was observed in S4.

When mutated to His or Cys, the residues R362 and A419C can be coordinated by metals (Zn²⁺ or Cd²⁺) in the open state of Shaker (17). Even though the nearby acidic residues E418 and E422 are located near the position A419, these residues are unlikely to be involved in the L366H:F370H Zn²⁺ coordination because, if the R362C highest point in the active state is near A419C, L366 and F370, respectively, one and two turns below residue R362, are therefore far from E418 and E422 (7). Residue E283 is one of the countercharge residues that stabilizes the open state, and it has been reported that they form salt bridges with residues R368 and R371 (5). L366H and F370H are oriented 200° apart in a α-helix or 240° in 3₁₀ helix structure from R368, and F370H is oriented 100° apart in a α-helix configuration or 120° in 3₁₀ from R371. Therefore, we cannot completely exclude the possibility that the coordination between L366H:F370H by Zn²⁺ would impair the ability of the R368 or R371 to coordinate with the countercharge E283. Nevertheless, such impairment would destabilize the open state of the channel, which would lead a rightward shift in the G-V curves, and the deactivation time constants would be faster. Only the fact that the deactivation time constant remains unaffected by Zn²⁺ is enough to make that assumption unlikely. We did not test the formation of the bridges with mutations at E283 because mutation at this residue dramatically influences the channel operation (5, 37). The lack of evidences of Zn²⁺ effects at a concentration of 20 μM on His:His spaced in i, i + 3 is consistent with the conclusion that when His:His are spaced in i, i + 4 in α-helix structure, it creates a better binding site for metals compared to when they are spaced in i, i + 3 (25). It has to be pointed out, however, that with the data presented here, it is not possible to exclude possible steric hindrances or influences from the lipids due to the coordination of His:His in presence of Zn²⁺. However, the lack of Zn²⁺ effects on the single mutants (L366H and F370H) and Zn²⁺ effects on the double mutant (L366H:F370H) supports the idea of a stabilization of the secondary structure of that S4 region in α-helix.

Collectively, our data suggest a hypothetical and simplified energy landscape for the transitions of the L366H:F370H channel (Fig. 7). We tested this hypothesis with a simple three-state model (resting, intermediate and active state; Fig. 7 A). To simulate the effect of Zn²⁺ in the energy landscape presented in Fig. 7 B, we simply divided the rates β and γ by a factor of 20. This makes the intermediate energy well deeper. Because we assume that a region of the S4 segment needs a transition to a 3₁₀-helical conformation to proceed to the active state, the physical justification of a deeper intermediate well in the presence of Zn²⁺ is that the α-helical stabilization by Zn²⁺ will make that transition less likely. All the rates used in this model are presented in Fig. S3. This simple model was able to reproduce qualitatively all the major experimental findings. The G-V curve is shifted to the right, and its maximal value measured at 1 s is decreased as the Zn²⁺ (q) probability of binding to a subunit is increased (Fig. 7 C). The model also reproduces the experimentally found increase of the slow component in the time course of the currents (Fig. 7 D) and the experimental result of no effect of Zn²⁺ on the deactivation time constants (Fig. 7, E and F). A complete description of the model and a complete set of activation currents generated with the model are presented in Fig. S3. Our simple model was successful to simulate only qualitatively the experimentally recorded currents in the presence and absence of Zn²⁺. It is possible that a more complete model could fit the data quantitatively, but such a model would need to include all the paths to activation and inactivation of the conduction pathway, such as the noncanonical coupling between the voltage sensors and the pore (38, 39).

Figure 7.

Model of His:His at S4 segment intra-subunit interaction in the presence of Zn²⁺ for L366H:F370H. (A) Three-state model for Shaker VSD. Resting and active states were assumed as α and intermediate state as 3₁₀-helix. The α and β are, respectively, the forward and backward rates for the transition from resting to intermediate state; the γ and δ are, respectively, the forward and backward rates for the transition from intermediate to active state. (B) Energy diagram at V_m = 0 mV for WT (black), L366:F370H (red), and Zn²⁺ effects on L366H:F370H (dashed red lines). Red dashed line shows that the intermediate state has the well deepened by Zn²⁺. (C) Simulated G-V curves are calculated using the peak of the currents presented in Fig. S3 for L366H:F370H, for several Zn²⁺ binding probability q = 0 (black squares), q = 0.15 (red circles), q = 0.23 (blue up triangle), and q = 0.33 (green up triangle). G-Vs curves were normalized by the maximal conductance (G) of the channel in the absence of Zn²⁺ (q = 0). Continuous lines over G-V curves are the best fittings of Eq. 2. The fitted parameters are as follows: V_1/2 = −10.5 ± 0.7 mV and z = 1.6 ± 0.1 for q = 0; V_1/2 = −5.2 ± 1.4 mV and z = 1.3 ± 0.1 for q = 0.15; V_1/2 = −2.7 ± 1.6 mV and z = 1.2 ± 0.1 for q = 0.23; and V_1/2 = 2.7 ± 2 mV and z = 1.0 ± 0.1 for q = 0.33. (D) Simulated activation ionic currents at 100 mV. (E) Simulated deactivation ionic currents at −150 mV after a 400 ms depolarizing pulse of 100 mV. (F) Two normalized simulated deactivation ionic currents are at different voltages −70 mV (left panel) and −150 mV (right panel). The color traces and q values used in (D–F) are the same as shown in (C). To see this figure in color, go online.

Conclusions

In summary, a His:His interaction indicates that the S4 segment is stabilized in α-helical conformation in the region between 366 and 370 of the S4 segment, slowing down activation kinetics and shifting the G-V curve to more depolarized voltages (∼10 mV). This result indicates that α-helical stabilization of this region of the S4 segment prevented a secondary structural short-lived transition. A plausible interpretation is that this intermediate state during activation in Shaker includes a short-lived 3₁₀-helical structure. Although our results cannot be extrapolated to another family of S4-based voltage sensor channels, we believe the strategy of His:His interactions using Zn²⁺ to coordinate them could help to probe transitions in other S4-based voltage sensors, which have been structurally and functionally well characterized, such as Na⁺ channels and other K⁺ channels.

Author Contributions

F.B., C.A.Z.B., and J.L.C.-d.-S. contributed to the conception and design of the project. C.A.Z.B. performed research and analyzed the data. C.A.Z.B. and J.L.C.-d.-S. wrote the manuscript with inputs from F.B.

Editor: Baron Chanda.