Membrane potential regulates the activity of voltage-dependent ion channels via specialized voltage-sensing modules, but the mechanisms involved in coupling voltage-sensor movement to pore opening remain unclear owing to a lack of resting state structures and robust methods to identify allosteric pathways. Here, using a newly developed interaction-energy analysis, we probe the interfaces of the voltage-sensing and pore modules in the Drosophila Shaker K+ channel. Our measurements reveal unexpectedly strong equilibrium gating interactions between contacts at the S4 and S5 helices in addition to those between S6 and the S4–S5 linker. Network analysis of MD trajectories shows that the voltage-sensor and pore motions are linked by two distinct pathways: a canonical pathway through the S4–S5 linker and a hitherto unknown pathway akin to rack-and-pinion coupling involving the S4 and S5 helices. Our findings highlight the central role of the S5 helix in electromechanical transduction in the voltage-gated ion channel (VGIC) superfamily.
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The authors thank K. Swartz and his colleagues for help with quantifying the expression of Shaker K+ channel mutants during the early stages of this project. We also thank D. E. Shaw and colleagues for generously sharing the trajectories of long MD simulations and J. Cowgill for help making Fig. 6. The calculations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at PDC Centre for High Performance Computing (PDC-HPC). This research was supported by funding from NIH to B.C. (NS081293, GM084140 and NS101723) and K.O. (T32-HL07936). B.C. is also supported by Romnes Faculty Fellowship (WARF).
Integrated supplementary information
(a and b) Average distance differences between the S4 helix and the S5 helix on the adjacent subunit. The differences represent the average distance of c-α atoms in the activated minus the average distance of c-α atoms in the resting state. (c and d) A cartoon representation showing two different types of motions that can occur during voltage-sensing displacement of S4. Panel C shows the downward deflection of S4 and the effect on residue distances between S4 and S5 whereas panel D show a reorientation of the angle between the long axis of S4 and S5 helix.
Charge per channel values (filled squares) for various mutants. The vertical dotted bars on each point represents a range corresponding to 95% confidence interval. For reference, the charge per channel for WT Shaker potassium channel which is 13.2 e0 is shown as orange dotted line.
Normalized G-V curves for WT is shown in grey in all the panels. Each panel shows a complete set of G-V curves corresponding to each of the single mutants and the corresponding double mutant. (a) R387A/F484A, (b) S376A/L382A, (c) S376A/Q383A, (d) V369A/V408A, (e) V369A/S412A, (f) I372A/I405A, (g) L409A/I372A, (h) V369A/S376A, (i) R387A/V369A. Error bars represent standard error of mean.
(a-d) Shortest pathways along the covariance network of the activated-state Kv1.2 simulation. Pathways begin at Arg 365 (R2; Shaker numbering) in each subunit and end at Val 474. The choice of sink residue (which subunit containing Val 474) was chosen based on the shortest path between each of the four possible Val (Supplementary Table 3). For consistency, colors for each subunit are the same as those in Fig. 2. For two of the subunits, the shortest path remains entirely within one subunit as it travels down S4, along the S4-S5 linker to Val 474 in S6 whereas for the other two, the shortest path goes from S4 to the neighboring S5 and moves down S5 to S6 rather than S6. (e-f) For subunits shown in panels C and D, the intersubunit pathways are dominant even when the sink residue is a V474 on the same subunit. (g-h) Betweenness is calculated in the activated state, using the Valine on the same subunit as the sink for subunits C and D. The intersubunit pathway is consistent when going to either the same or adjacent subunit valine. (i-l) Shortest pathways along the covariance network for the resting-state simulations. The source residue is Arg 365 in each subunit and V474. The shortest path remains entirely within one subunit as it travels down S4, along the S4-S5 linker to Val 474 in S6. In the resting state, unlike the activated state, there is no intersubunit pathway. (m-n) Experimentally determined long range interactions are shown along the optimal pathway in subunit A in the activated state and subunit D in the resting state. It is possible that these residues show interactions while being distant from one another because they are on pathways allosterically linking the VSD to the pore domain.
All the single and the double mutants (red) are shown as noted in the legends. The mutants are plotted along with the WT (black) collected from the same batch of oocytes in order to minimize batch to batch variations. Grey lines define the two-sided bounds with 95% confidence intervals.
The sequence alignment is used to convert Kv 1.2/2.1 numbering to that of Shaker K+ channel. Homologous residues are highlighted in black, conservative mutations in gray and distinct mutations in white.
Supplementary Figures 1–6 and Supplementary Tables 1–3
Propagation of voltage-sensor movement. The deactivation of the voltage sensor (blue) is concomitant with downward movement of the neighboring S5, which ultimately results in straightening of the C terminus of the S6 helix