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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Long, S. B., Campbell, E. B. & Mackinnon, R. Crystal structure of a mammalian voltage-dependent Shaker family K+channel. Science 309, 897–903 (2005).
Männikkö, R., Elinder, F. & Larsson, H. P. Voltage-sensing mechanism is conserved among ion channels gated by opposite voltages. Nature 419, 837–841 (2002).
Larsson, H. P. The search is on for the voltage sensor-to-gate coupling. J. Gen. Physiol. 120, 475–481 (2002).
Lu, Z., Klem, A. M. & Ramu, Y. Ion conduction pore is conserved among potassium channels. Nature 413, 809–813 (2001).
Lu, Z., Klem, A. M. & Ramu, Y. Coupling between voltage sensors and activation gate in voltage-gated K+ channels. J. Gen. Physiol. 120, 663–676 (2002).
Jiang, Y. et al. Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522 (2002).
Guo, J. et al. Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana. Nature 531, 196–201 (2016).
Randich, A. M., Cuello, L. G., Wanderling, S. S. & Perozo, E. Biochemical and structural analysis of the hyperpolarization-activated K+ channel MVP. Biochemistry. 53, 1627–1636 (2014).
Blunck, R. & Batulan, Z. Mechanism of electromechanical coupling in voltage-gated potassium channels. Front. Pharmacol. 3, 166 (2012).
Hoshi, T., Pantazis, A. & Olcese, R. Transduction of voltage and Ca2+ signals by Slo1 BK channels. Physiology (Bethesda). 28, 172–189 (2013).
Lörinczi, É. et al. Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains. Nat. Commun. 6, 6672 (2015).
Lee, C. H. & MacKinnon, R. Structures of the human HCN1 hyperpolarization-activated channel. Cell 168, 111–120.e11 (2017).
Payandeh, J., Gamal El-Din, T. M., Scheuer, T., Zheng, N. & Catterall, W. A. Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. Nature 486, 135–139 (2012).
Whicher, J. R. & MacKinnon, R. Structure of the voltage-gated K+ channel Eag1 reveals an alternative voltage sensing mechanism. Science 353, 664–669 (2016).
Payandeh, J., Scheuer, T., Zheng, N. & Catterall, W. A. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011).
Shen, H. et al. Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution. Science 355, eaal4326 (2017).
Zhang, X. et al. Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel. Nature 486, 130–134 (2012).
Tsai, C. J. et al. Two alternative conformations of a voltage-gated sodium channel. J. Mol. Biol. 425, 4074–4088 (2013).
Long, S. B., Campbell, E. B. & Mackinnon, R. Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309, 903–908 (2005).
Wu, J. et al. Structure of the voltage-gated calcium channel Ca(v)1.1 at 3.6 Å resolution. Nature 537, 191–196 (2016).
Jensen, M. O. et al. Mechanism of voltage gating in potassium channels. Science 336, 229–233 (2012).
Vargas, E., Bezanilla, F. & Roux, B. In search of a consensus model of the resting state of a voltage-sensing domain. Neuron. 72, 713–720 (2011).
Hackos, D. H., Chang, T. H. & Swartz, K. J. Scanning the intracellular S6 activation gate in the shaker K + channel. J. Gen. Physiol. 119, 521–531 (2002).
Ledwell, J. L. A. & Aldrich, R. W. Mutations in the S4 region isolate the final voltage-dependent cooperative step in potassium channel activation. J. Gen. Physiol. 113, 389–414 (1999).
Soler-Llavina, G. J., Chang, T. H. & Swartz, K. J. Functional interactions at the interface between voltage-sensing and pore domains in the Shaker Kv channel. Neuron. 52, 623–634 (2006).
Chowdhury, S., Haehnel, B. M. & Chanda, B. A self-consistent approach for determining pairwise interactions that underlie channel activation. J. Gen. Physiol. 144, 441–455 (2014).
Chowdhury, S., Haehnel, B. M. & Chanda, B. Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels. J. Gen. Physiol. 144, 457–467 (2014).
Chowdhury, S. & Chanda, B. Perspectives on: conformational coupling in ion channels: thermodynamics of electromechanical coupling in voltage-gated ion channels. J. Gen. Physiol. 140, 613–623 (2012).
Miller, C. Model-free free energy for voltage-gated channels. J. Gen. Physiol. 139, 1–2 (2012).
Yifrach, O. No model in mind: a model-free approach for studying ion channel gating. J. Gen. Physiol. 141, 3–9 (2013).
Muroi, Y., Arcisio-Miranda, M., Chowdhury, S. & Chanda, B. Molecular determinants of coupling between the domain III voltage sensor and pore of a sodium channel. Nat. Struct. Mol. Biol. 17, 230–237 (2010).
Batulan, Z., Haddad, G. A. & Blunck, R. An intersubunit interaction between S4-S5 linker and S6 is responsible for the slow off-gating component in Shaker K+ channels. J. Biol. Chem. 285, 14005–14019 (2010).
Labro, A. J. et al. Kv channel gating requires a compatible S4-S5 linker and bottom part of S6, constrained by non-interacting residues. J. Gen. Physiol. 132, 667–680 (2008).
Chen, J., Mitcheson, J. S., Tristani-Firouzi, M., Lin, M. & Sanguinetti, M. C. The S4-S5 linker couples voltage sensing and activation of pacemaker channels. Proc. Natl Acad. Sci. USA 98, 11277–11282 (2001).
Pless, S. A. & Ahern, C. A. Introduction: applying chemical biology to ion channels. Adv. Exp. Med. Biol. 869, 1–4 (2015).
Schoppa, N. E. & Sigworth, F. J. Activation of Shaker potassium channels. II. Kinetics of the V2 mutant channel. J. Gen. Physiol. 111, 295–311 (1998).
Schoppa, N. E. & Sigworth, F. J. Activation of Shaker potassium channels. III. An activation gating model for wild-type and V2 mutant channels. J. Gen. Physiol. 111, 313–342 (1998).
McCormack, K., Lin, L. & Sigworth, F. J. Substitution of a hydrophobic residue alters the conformational stability of Shaker K + channels during gating and assembly. Biophys. J. 65, 1740–1748 (1993).
Smith-Maxwell, C. J., Ledwell, J. L. & Aldrich, R. W. Uncharged S4 residues and cooperativity in voltage-dependent potassium channel activation. J. Gen. Physiol. 111, 421–439 (1998).
Smith-Maxwell, C. J., Ledwell, J. L. & Aldrich, R. W. Role of the S4 in cooperativity of voltage-dependent potassium channel activation. J. Gen. Physiol. 111, 399–420 (1998).
Li-Smerin, Y., Hackos, D. H. & Swartz, K. J. A localized interaction surface for voltage-sensing domains on the pore domain of a K + channel. Neuron. 25, 411–423 (2000).
Hong, K. H. & Miller, C. The lipid-protein interface of a Shaker K+ channel. J. Gen. Physiol. 115, 51–58 (2000).
Sadovsky, E. & Yifrach, O. Principles underlying energetic coupling along an allosteric communication trajectory of a voltage-activated K + channel. Proc. Natl Acad. Sci. USA 104, 19813–19818 (2007).
Yifrach, O. & MacKinnon, R. Energetics of pore opening in a voltage-gated K( + ) channel. Cell 111, 231–239 (2002).
del Camino, D., Holmgren, M., Liu, Y. & Yellen, G. Blocker protection in the pore of a voltage-gated K + channel and its structural implications. Nature 403, 321–325 (2000).
Swartz, K. J. Structure and anticipatory movements of the S6 gate in Kv channels. J. Gen. Physiol. 126, 413–417 (2005).
Jensen, M. O. et al. Principles of conduction and hydrophobic gating in K + channels. Proc. Natl Acad. Sci. USA 107, 5833–5838 (2010).
Swartz, K. J. Sensing voltage across lipid membranes. Nature 456, 891–897 (2008).
Arrigoni, C. et al. The voltage-sensing domain of a phosphatase gates the pore of a potassium channel. J. Gen. Physiol. 141, 389–395 (2013).
Wang, W. & MacKinnon, R. Cryo-EM structure of the open human ether-a-go-go-related K + channel hERG. Cell 169, 422–430.e10 (2017).
Sethi, A., Eargle, J., Black, A. A. & Luthey-Schulten, Z. Dynamical networks in tRNA:protein complexes. Proc. Natl Acad. Sci. USA 106, 6620–6625 (2009).
LeVine, M. V. & Weinstein, H. NbIT–a new information theory-based analysis of allosteric mechanisms reveals residues that underlie function in the leucine transporter LeuT. PLoS. Comput. Biol. 10, e1003603 (2014).
Hoshi, T., Zagotta, W. N. & Aldrich, R. W. Shaker potassium channel gating. I: Transitions near the open state. J. Gen. Physiol. 103, 249–278 (1994).
Perozo, E., MacKinnon, R., Bezanilla, F. & Stefani, E. Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K + channels. Neuron. 11, 353–358 (1993).
Muroi, Y. & Chanda, B. Local anesthetics disrupt energetic coupling between the voltage-sensing segments of a sodium channel. J. Gen. Physiol. 133, 1–15 (2009).
Gamal El-Din, T. M., Grögler, D., Lehmann, C., Heldstab, H. & Greeff, N. G. More gating charges are needed to open a Shaker K + channel than are needed to open an rBIIA Na + channel. Biophys. J. 95, 1165–1175 (2008).
Seoh, S. A., Sigg, D., Papazian, D. M. & Bezanilla, F. Voltage-sensing residues in the S2 and S4 segments of the Shaker K+channel. Neuron 16, 1159–1167 (1996).
Aggarwal, S. K. M. & MacKinnon, R. Contribution of the S4 segment to gating charge in the Shaker K+channel. Neuron 16, 1169–1177 (1996).
Schoppa, N. E., McCormack, K., Tanouye, M. A. & Sigworth, F. J. The size of gating charge in wild-type and mutant Shaker potassium channels. Science 255, 1712–1715 (1992).
Eargle, J. & Luthey-Schulten, Z. NetworkView: 3D display and analysis of protein·RNA interaction networks. Bioinformatics 28, 3000–3001 (2012).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).
Glykos, N. M. Software news and updates. Carma: a molecular dynamics analysis program. J. Comput. Chem. 27, 1765–1768 (2006).
Hagberg, A. A., Schult, D. A. & Swart, P. J. Exploring network structure, dynamics, and function using NetworkX. In Proc. 7th Python in Science Conference (SciPy 2008) (Eds Varoquaux, G., Vaught, T. & Millman, J.) 11–15 (2008).
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).
The authors declare no competing interests.
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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
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Fernández-Mariño, A.I., Harpole, T.J., Oelstrom, K. et al. Gating interaction maps reveal a noncanonical electromechanical coupling mode in the Shaker K+ channel. Nat Struct Mol Biol 25, 320–326 (2018). https://doi.org/10.1038/s41594-018-0047-3
Nature Communications (2020)
Nature Communications (2019)