KirBac1.1: it's an inward rectifying potassium channel.
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 (86)Rb(+) 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 Ba(2+), 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 PIP(2), consistent with (86)Rb(+) flux experiments, and reversibly inhibited by short-chain di-c8-PIP(2). 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.
Cheng
Wayland W L
WW
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Enkvetchakul
Decha
D
Nichols
Colin G
CG
D016957:Burkholderia pseudomallei / Q000502:physiology; D015640:Ion Channel Gating; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D018408:Patch-Clamp Techniques; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011487:Protein Conformation; D013096:Spermine / Q000494:pharmacology
D008081:Liposomes; D024661:Potassium Channels, Inwardly Rectifying; D013096:Spermine