Abstract
The bacterial potassium channel KcsA is gated by pH, opening for conduction under acidic conditions. Molecular determinants responsible for this effect have been identified at the extracellular selectivity filter, at the membrane–cytoplasm interface (TM2 gate), and in the cytoplasmic C-terminal domain (CTD), an amphiphilic four-helix bundle mediated by hydrophobic and electrostatic interactions. Here we have employed NMR and EPR to provide a structural view of the pH-induced open-to-closed CTD transition. KcsA was embedded in lipoprotein nanodiscs (LPNs), selectively methyl-protonated at Leu/Val residues to allow observation of both states by NMR, and spin-labeled for the purposes of EPR studies. We observed a pHinduced structural change between an associated structured CTD at neutral pH and a dissociated flexible CTD at acidic pH, with a transition in the 5.0–5.5 range, consistent with a stabilization of the CTD by channel architecture. A double mutant constitutively open at the TM2 gate exhibited reduced stability of associated CTD, as indicated by weaker spin–spin interactions, a shift to higher transition pH values, and a tenfold reduction in the population of the associated “closed” channels. We extended these findings for isolated CTD-derived peptides to full-length KcsA and have established a contribution of the CTD to KcsA pH-controlled gating, which exhibits a strong correlation with the state of the proximal TM2 gate.
Original language | English |
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Pages (from-to) | 813-821 |
Number of pages | 9 |
Journal | ChemBioChem |
Volume | 20 |
Issue number | 6 |
DOIs | |
State | Published - 15 Mar 2019 |
Bibliographical note
Publisher Copyright:© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Funding
We thank Drs. Hugo Gottlieb and Keren Keinan-Adamsky for spectrometer assistance and Israel Tabakman for technical assistance. We are grateful to Dr. Guy Kamnesky (Technion–Israel Institute of Technology, Haifa, Israel) for preparation of the KcsA(128–160) sample. The MSP1D1-encoding plasmid was generously provided by Prof. Franz Hagn (Technische Universitat München). Financial support by the Israel Science Foundation (award 1088/16) and the Binational Israel-US Foundation (award 2013185) is gratefully acknowledged. Establishment of the 700 MHz spectrometer system was supported by Fund#cion Adar and a Converging Technologies award. J.H.C. acknowledges the support of the Christians for Israel Chair for Medical Research. We thank Drs. Hugo Gottlieb and Keren Keinan-Adamsky for spectrometer assistance and Israel Tabakman for technical assistance. We are grateful to Dr. Guy Kamnesky (Technion?Israel Institute of Technology, Haifa, Israel) for preparation of the KcsA(128?160) sample. The MSP1D1-encoding plasmid was generously provided by Prof. Franz Hagn (Technische Universitat M?nchen). Financial support by the Israel Science Foundation (award 1088/16) and the Binational Israel-US Foundation (award 2013185) is gratefully acknowledged. Establishment of the 700 MHz spectrometer system was supported by Fund?cion Adar and a Converging Technologies award. J.H.C. acknowledges the support of the Christians for Israel Chair for Medical Research.
Funders | Funder number |
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Binational Israel-US Foundation | 2013185 |
Technion?Israel Institute of Technology, Haifa, Israel | |
Technische Universitat M?nchen | |
Israel Science Foundation | 1088/16 |
Keywords
- EPR spectroscopy
- KcsA
- NMR spectroscopy
- ion channels
- lipoprotein nanodiscs
- pH gating