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Gating interaction maps reveal a noncanonical electromechanical coupling mode in the Shaker K+ channel

Abstract

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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Fig. 1: Interfacial regions and residues tested for electromechanical coupling.
Fig. 2: Interaction-energy analysis of residues in the intracellular gating interface.
Fig. 3: Interaction-energy analysis of residues in the transmembrane gating interface.
Fig. 4: Long-distance interactions between the S4 and the S4–S5 linker of the same subunit.
Fig. 5: Residue betweenness for pathways between S4 and S6 in the activated (open) state.
Fig. 6: Schematic showing the two potential modes of electromechanical coupling in a prototypical potassium channel.

References

  1. 1.

    Long, S. B., Campbell, E. B. & Mackinnon, R. Crystal structure of a mammalian voltage-dependent Shaker family K+channel. Science 309, 897–903 (2005).

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Männikkö, R., Elinder, F. & Larsson, H. P. Voltage-sensing mechanism is conserved among ion channels gated by opposite voltages. Nature 419, 837–841 (2002).

    Article  PubMed  Google Scholar 

  3. 3.

    Larsson, H. P. The search is on for the voltage sensor-to-gate coupling. J. Gen. Physiol. 120, 475–481 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Lu, Z., Klem, A. M. & Ramu, Y. Ion conduction pore is conserved among potassium channels. Nature 413, 809–813 (2001).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Jiang, Y. et al. Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522 (2002).

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Guo, J. et al. Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana. Nature 531, 196–201 (2016).

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Blunck, R. & Batulan, Z. Mechanism of electromechanical coupling in voltage-gated potassium channels. Front. Pharmacol. 3, 166 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Hoshi, T., Pantazis, A. & Olcese, R. Transduction of voltage and Ca2+ signals by Slo1 BK channels. Physiology (Bethesda). 28, 172–189 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    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).

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Lee, C. H. & MacKinnon, R. Structures of the human HCN1 hyperpolarization-activated channel. Cell 168, 111–120.e11 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Whicher, J. R. & MacKinnon, R. Structure of the voltage-gated K+ channel Eag1 reveals an alternative voltage sensing mechanism. Science 353, 664–669 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Payandeh, J., Scheuer, T., Zheng, N. & Catterall, W. A. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Shen, H. et al. Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution. Science 355, eaal4326 (2017).

    Article  PubMed  Google Scholar 

  17. 17.

    Zhang, X. et al. Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel. Nature 486, 130–134 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Tsai, C. J. et al. Two alternative conformations of a voltage-gated sodium channel. J. Mol. Biol. 425, 4074–4088 (2013).

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Long, S. B., Campbell, E. B. & Mackinnon, R. Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309, 903–908 (2005).

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    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).

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Jensen, M. O. et al. Mechanism of voltage gating in potassium channels. Science 336, 229–233 (2012).

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    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).

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Miller, C. Model-free free energy for voltage-gated channels. J. Gen. Physiol. 139, 1–2 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Yifrach, O. No model in mind: a model-free approach for studying ion channel gating. J. Gen. Physiol. 141, 3–9 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Pless, S. A. & Ahern, C. A. Introduction: applying chemical biology to ion channels. Adv. Exp. Med. Biol. 869, 1–4 (2015).

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    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).

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Hong, K. H. & Miller, C. The lipid-protein interface of a Shaker K+ channel. J. Gen. Physiol. 115, 51–58 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Yifrach, O. & MacKinnon, R. Energetics of pore opening in a voltage-gated K( + ) channel. Cell 111, 231–239 (2002).

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    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).

    Article  PubMed  Google Scholar 

  46. 46.

    Swartz, K. J. Structure and anticipatory movements of the S6 gate in Kv channels. J. Gen. Physiol. 126, 413–417 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Jensen, M. O. et al. Principles of conduction and hydrophobic gating in K + channels. Proc. Natl Acad. Sci. USA 107, 5833–5838 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Swartz, K. J. Sensing voltage across lipid membranes. Nature 456, 891–897 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    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).

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    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).

    Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    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).

    CAS  Article  PubMed  Google Scholar 

  54. 54.

    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).

    CAS  Article  PubMed  Google Scholar 

  55. 55.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56.

    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).

    Article  PubMed  PubMed Central  Google Scholar 

  57. 57.

    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).

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Aggarwal, S. K. M. & MacKinnon, R. Contribution of the S4 segment to gating charge in the Shaker K+channel. Neuron 16, 1169–1177 (1996).

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    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).

    CAS  Article  PubMed  Google Scholar 

  60. 60.

    Eargle, J. & Luthey-Schulten, Z. NetworkView: 3D display and analysis of protein·RNA interaction networks. Bioinformatics 28, 3000–3001 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).

    CAS  Article  PubMed  Google Scholar 

  62. 62.

    Glykos, N. M. Software news and updates. Carma: a molecular dynamics analysis program. J. Comput. Chem. 27, 1765–1768 (2006).

    CAS  Article  PubMed  Google Scholar 

  63. 63.

    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).

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Acknowledgements

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).

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A.I.F.-M. contributed to design, acquisition and analysis of experimental data and writing the manuscript. T.J.H. carried out the network analyses, analyzed and interpreted the simulation data and contributed to writing the manuscript. K.O. contributed to design and acquisition of the experimental data. L.D. designed the network analyses, analyzed and interpreted data and contributed to writing the manuscript. B.C. conceived the project, designed experiments, interpreted data and wrote the manuscript.

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Correspondence to Baron Chanda.

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Integrated supplementary information

Supplementary Figure 1 Relative molecular motions between S4 and S5 upon voltage activation.

(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.

Supplementary Figure 2 Experimentally determined charge per channel for each mutant.

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.

Supplementary Figure 3 G–V curves for the mutants at the gating interface.

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.

Supplementary Figure 4 Network analysis of the Kv1.2 simulations.

(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.

Supplementary Figure 5 Qmax–fluorescence relationships for each mutant.

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.

Supplementary Figure 6 Sequence alignment between Shaker and Kv 1.2/2.1.

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.

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Supplementary Figures 1–6 and Supplementary Tables 1–3

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Supplementary Video 1

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

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