Investigation of a KcsA Cytoplasmic pH Gate in Lipoprotein Nanodiscs

Arwa Qasim, Inbal Sher, Orel Hirschhorn, Hadassa Shaked, Zena Qasem, Sharon Ruthstein, Jordan H. Chill

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

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 languageEnglish
Pages (from-to)813-821
Number of pages9
JournalChemBioChem
Volume20
Issue number6
DOIs
StatePublished - 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.

FundersFunder number
Binational Israel-US Foundation2013185
Technion?Israel Institute of Technology, Haifa, Israel
Technische Universitat M?nchen
Israel Science Foundation1088/16

    Keywords

    • EPR spectroscopy
    • KcsA
    • NMR spectroscopy
    • ion channels
    • lipoprotein nanodiscs
    • pH gating

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