Lewis A et al. (2008), Atomic constraints between the voltage sensor a...

XB-ART-38161

J Gen Physiol 2008 Jun 01;1316:549-61. doi: 10.1085/jgp.200809962.

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Atomic constraints between the voltage sensor and the pore domain in a voltage-gated K+ channel of known structure.

Lewis A , Jogini V , Blachowicz L , Lainé M , Roux B .

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In voltage-gated K(+) channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn(2+) or Cd(2+) with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by approximately 7-8 A and rotated counterclockwise by 37 degrees along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.

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Species referenced: Xenopus
Genes referenced: kcna2 kcnb1 tbx2 tyro3

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	Figure 1. Comparison of amino acid sequence of Shaker and Kv1.2 in the region of the S4 and S5 segments. (A) A model describing the membrane topology of a Kv1.2 subunit showing transmembrane segments S1–S6 and the reentrant P loop. The approximate positions of residues R294 of S4 segment and A351 of the S5 pore domain are indicated. (B) Alignment of S4–S5 regions of Kv1.2, Shaker, KvAP, and the recently crystallized chimera of Kv1.2-Kv2.1. The black line indicates the S4 and S5 transmembrane segments and the asterisks denote the position of residues R294 and A351 of Kv1.2. Highlighted in blue and red are basic and acidic residues, respectively.
	Figure 2. R294H-A351H forms a high affinity Zn2+ binding site that reduces current magnitude. (A) Representative current traces from an oocyte expressing Kv1.2-R294H-A351H channels recorded in the absence (left) and presence (right) of 1 μM Zn2+, using the protocol outlined in the Materials and methods. Black arrowheads indicate where steady-state current was measured to generate I-V and G-V relationships. (B) Mean current–voltage relationships from oocytes expressing Kv1.2-R294H-A351H channels recorded in the absence (solid squares, n = 8) and presence (open circles, n = 8) of 1 μM Zn2+. (C) Normalized G-V curves for Kv1.2-R294H-A351H obtained in the absence (black squares, n = 8) and presence of various concentrations of Zn2+; 10 nM (gray triangles, n = 7), 100 nM (open diamonds, n = 7), and 1 μM (open circles, n = 8) showing dose-dependent reduction in conductance with increasing concentrations of extracellular Zn2+. To demonstrate loss of current with Zn2+ application, conductance (G) values obtained in either the absence or presence of Zn2+ were normalized to the maximal G value obtained in the absence of Zn2+. Data for control (no Zn2+) and 10 nM Zn2+ were fitted with a single Boltzmann function, shown as solid black and gray curves respectively. (D and E) Normalized G-V curves obtained in the absence (solid squares) and presence (open circles) of 1 μM Zn2+ for (D) Kv1.2-R294H (n = 9) and (E) Kv1.2-A351H (n = 8). Single mutants showed no loss of current magnitude with application of 1 μM Zn2+; hence G values were normalized to the maximal G in each experimental condition (absence or presence of Zn2+).
	Figure 3. Cd2+ can form a metal bridge between R294H and A351H. (A) Representative current traces from an oocyte expressing Kv1.2-R294H-A351H channels recorded in the absence (left) and presence (right) of 1 μM Cd2+. Black arrowheads indicate where steady-state current was measured to generate I-V and G-V relationships. (B) Mean current–voltage relationships from oocytes expressing Kv1.2-R294H-A351H channels recorded in the absence (solid squares, n = 6) and presence (open circles, n = 6) of 1 μM Cd2+. (C) Normalized G-V relationships from oocytes expressing Kv1.2-R294H-A351H channels recorded in the absence (solid squares, n = 6) and presence (open circles, n = 6) of 1 μM Cd2+.
	Figure 4. Metal ion binding to R294H-A351H stabilizes the activated state after neutralization of acidic residues D352 and E353. (A) Representative current traces from an oocyte expressing Kv1.2-R294H-A351H-D352G-E353S channels recorded in the absence (left) and presence (right) of 1 μM Zn2+, using the protocol outlined in the Materials and methods. (B) Normalized G-V relationships from oocytes expressing Kv1.2-R294H-A351H-D352G-E353S channels recorded in the absence (solid squares, n = 9) and presence (open circles, n = 9) of 1 μM Zn2+. (C) A plot of ΔV1/2 versus Zn2+ concentration in oocytes expressing Kv1.2-R294H-A351H-D352G-E353S channels, where ΔV1/2 is the difference between the fitted V1/2 values obtained in the absence and presence of varying concentrations of Zn2+. The data were fitted with a hyperbolic function (solid curve) with a half-maximal concentration of 0.36 ± 0.18 μM (n = 4–8). (D and E) Normalized G-V curves obtained in the absence (solid squares) and presence (open circles) of 1 μM Zn2+ for (D) Kv1.2-R294H-D352G-E353S (n = 7) and (E) Kv1.2-A351H-D352G-E353S (n = 7).
	Figure 5. Metal bridge formation slows channel closure signifying stabilization of the open-activated channel conformation. (A) Representative current traces from an oocyte expressing Kv1.2-R294H-A351H-D352G-E353S channels recorded in the absence (left) and presence (right) of 1 μM Zn2+. Bath solution contained 50 mM K+ in order to visualize inward tail currents. (B) Plot of time constant for deactivation versus voltage for channels recorded in the absence (solid squares) or presence (open circles) of 1 μM Zn2+ (n = 7 each). Tail currents were fitted with a single exponential and the time constant of deactivation (τ, ms) plotted on a log scale against voltage.
	Figure 6. R294H-D352H forms a lower affinity Zn2+ site but is still able to promote the activated conformational state. (A and B) Normalized G-V relationships from oocytes expressing (A) Kv1.2-R294H-D352H (n = 7) and (B) Kv1.2-D352H (n = 7) channels recorded in the absence (solid squares) and presence (open circles) of 1 μM Zn2+. (C) A plot of ΔV1/2 versus Zn2+ concentration in oocytes expressing Kv1.2-R294H-D352H channels, where ΔV1/2 is the difference between the fitted V1/2 values obtained in the absence and presence of varying concentrations of Zn2+. The data were fitted with a hyperbolic function (solid curve) with a half-maximal concentration of 470 nM (n = 5–10).
	Figure 7. Comparison of the Kv1.2 crystal structure (Long et al., 2005) with instantaneous configurations taken from the MD simulations of the wild-type channel and mutant channels. (A) R294H-A351H-D352G-E353S (side and top view). (B) R294H-A351H (top view). (C) R294H-D352H (top view). Crystal structure is shown in gray. Mutant channel is represented as following: S1-cyan, S2-yellow, S3-red, S4-blue, pore-green, and Zn2+-violet. All the mutant structures were taken from the last snapshot of the MD trajectories. (See online supplemental material for the coordinates of the open activated Kv1.2 model obtained from R294H-A351H-D352G-E353S simulation in the presence of metal bridge).
	Figure 8. Position of the first gating charge along the S4 segment (R294) in the open-activated conformation of Kv1.2 channel deduced from MD. The mesh plots encompass the volume occupied 60% of the time by the Cβ of residue 294 during the different MD simulations. The data shown includes the wild-type Kv1.2 channel (A, orange mesh), the Kv1.2 channel with R294H-A351H Zn2+ bridge (B, gray mesh), the Kv1.2 channel with the R294H-D352H Zn2+ bridge (C, cyan mesh), and the Kv1.2 channel with the R294H-D352H Zn2+ bridge and the substitution D352G-E353S (D, blue mesh). On the left is a side view from membrane and on the right is a side view from the pore. As a reference, the S4 segment (sky blue ribbon) and the pore domain (pale green ribbon) from the crystal structure of the Kv1.2 channel (PDB id 2A79) are shown. Three K+ ions are shown in the pore as orange spheres (left). The Cβ of residue 294 and the Cβ of residue 351 from the Kv1.2 x-ray structure are shown as red and magenta spheres, respectively. The Cβ of the residue at the corresponding position in the x-ray structure of the Kv1.2-Kv2.1 chimera PDB id 2R9R (Q290) is shown as a green sphere (assuming the same position for the pore domain). To record the probability density of the Cβ of residue 294 from the MD, the pore domain (S5–S6) was oriented with respect to the x-ray structure for every snapshot, followed by increments of 90° rotations to superimpose the voltage sensor modules of the four subunits (Jogini and Roux, 2007), yielding four data points per snapshot. For the wild-type channel, one snapshot every 5 ps is included (total 20,000 points), for each mutant one snapshot every 2 ps from two trajectories is included (total 10,000 points).

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase