Investigation of a KcsA Cytoplasmic pH Gate in Lipoprotein Nanodiscs. (5th February 2019)

• Record Type: Journal Article
• Authors: Qasim, Arwa; Sher, Inbal; Hirschhorn, Orel; Shaked, Hadassa; Qasem, Zena; Ruthstein, Sharon; Chill, Jordan H.
• Is Part Of: Chembiochem. Volume 20:Number 6(2019)
• Page Start: 813
• Page End: 821
• Publication Date: 2019-02-05
• Subjects: EPR spectroscopy -- ion channels -- KcsA -- lipoprotein nanodiscs -- NMR spectroscopy -- pH gating; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201800627
• Languages: English
• ISSNs: 1439-4227

Abstract

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. Abstract : Watching the gate "swing" open : NMR and EPR were employed to follow pH‐induced changes in the cytoplasmic C‐terminal gate of the KcsA potassium channel. By embedding the full‐length channel in lipoprotein nanodiscs we could observe open and closed states and investigate the cross‐interaction between the C‐terminal and membrane gates of the channel.