title: A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore.
abstract: Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4-S5 linker). However, our recent work on channels disrupted in the S4-S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of KV10.1 revealed that the S4-S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use "split" channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in KV10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4-S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4-S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism.
authors.affiliation: Oncophysiology Group, Max Planck Institute for Experimental Medicine, 37075 Göttingen, Germany.
authors.forename: Adam P
authors.identifier: http://orcid.org/0000-0003-4814-4532
authors.initials: AP
authors.lastname: Tomczak
authors.affiliation: Oncophysiology Group, Max Planck Institute for Experimental Medicine, 37075 Göttingen, Germany.
authors.forename: Jorge
authors.initials: J
authors.lastname: Fernández-Trillo
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.|Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Shashank
authors.initials: S
authors.lastname: Bharill
authors.affiliation: Department of Biophysics and Cell Biology, University of Debrecen, 4032 Debrecen, Hungary.|MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, University of Debrecen, 4032 Debrecen, Hungary.
authors.forename: Ferenc
authors.identifier: http://orcid.org/0000-0002-9549-6066
authors.initials: F
authors.lastname: Papp
authors.affiliation: Department of Biophysics and Cell Biology, University of Debrecen, 4032 Debrecen, Hungary.|MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, University of Debrecen, 4032 Debrecen, Hungary.
authors.forename: Gyorgy
authors.initials: G
authors.lastname: Panyi
authors.affiliation: Department of Molecular Biology of Neuronal Signals, Max Planck Institute for Experimental Medicine, 37075 Göttingen, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Stühmer
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.|Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Ehud Y
authors.initials: EY
authors.lastname: Isacoff
authors.affiliation: Oncophysiology Group, Max Planck Institute for Experimental Medicine, 37075 Göttingen, Germany Pardo@em.mpg.de.
authors.forename: Luis A
authors.identifier: http://orcid.org/0000-0003-1375-4349
authors.initials: LA
authors.lastname: Pardo
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating; D008564:Membrane Potentials; D000072417:Protein Domains; D014981:Xenopus
chemical_list: D051638:Ether-A-Go-Go Potassium Channels