KirBac1.1: It's an Inward Rectifying Potassium Channel
Open Access
- 9 February 2009
- journal article
- Published by Rockefeller University Press in The Journal of general physiology
- Vol. 133 (3) , 295-305
- https://doi.org/10.1085/jgp.200810125
Abstract
KirBac1.1 is a prokaryotic homologue of eukaryotic inward rectifier potassium (Kir) channels. The crystal structure of KirBac1.1 and related KirBac3.1 have now been used extensively to generate in silico models of eukaryotic Kir channels, but functional analysis has been limited to 86Rb+ flux experiments and bacteria or yeast complementation screens, and no voltage clamp analysis has been available. We have expressed pure full-length His-tagged KirBac1.1 protein in Escherichia coli and obtained voltage clamp recordings of recombinant channel activity in excised membrane patches from giant liposomes. Macroscopic currents of wild-type KirBac1.1 are K+ selective and spermine insensitive, but blocked by Ba2+, similar to “weakly rectifying” eukaryotic Kir1.1 and Kir6.2 channels. The introduction of a negative charge at a pore-lining residue, I138D, generates high spermine sensitivity, similar to that resulting from the introduction of a negative charge at the equivalent position in Kir1.1 or Kir6.2. KirBac1.1 currents are also inhibited by PIP2, consistent with 86Rb+ flux experiments, and reversibly inhibited by short-chain di-c8-PIP2. At the single-channel level, KirBac1.1 channels show numerous conductance states with two predominant conductances (15 pS and 32 pS at −100 mV) and marked variability in gating kinetics, similar to the behavior of KcsA in recombinant liposomes. The successful patch clamping of KirBac1.1 confirms that this prokaryotic channel behaves as a bona fide Kir channel and opens the way for combined biochemical, structural, and electrophysiological analysis of a tractable model Kir channel, as has been successfully achieved for the archetypal K+ channel KcsA.Keywords
This publication has 60 references indexed in Scilit:
- Differential Roles of Blocking Ions in KirBac1.1 Tetramer StabilityJournal of Biological Chemistry, 2009
- Interaction of Anesthetics with Open and Closed Conformations of a Potassium Channel Studied via Molecular Dynamics and Normal Mode AnalysisBiophysical Journal, 2008
- Pore-opening mechanism of the nicotinic acetylcholine receptor evinced by proton transferNature Structural & Molecular Biology, 2008
- A Quantitative Description of KcsA Gating II: Single-Channel CurrentsThe Journal of general physiology, 2007
- A Quantitative Description of KcsA Gating I: Macroscopic CurrentsThe Journal of general physiology, 2007
- Crystal structure of a Kir3.1-prokaryotic Kir channel chimeraThe EMBO Journal, 2007
- Control of Inward Rectifier K Channel Activity by Lipid Tethering of Cytoplasmic DomainsThe Journal of general physiology, 2007
- Identification of the PIP2-binding site on Kir6.2 by molecular modelling and functional analysisThe EMBO Journal, 2007
- The Polyamine Binding Site in Inward Rectifier K+ ChannelsThe Journal of general physiology, 2006
- Single Streptomyces lividans K+ ChannelsThe Journal of general physiology, 1999