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KirBac1.1: it's an inward rectifying potassium channel.

Cheng WW, Enkvetchakul D, Nichols CG - J. Gen. Physiol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

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 (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.

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Single-channel recordings show significant gating heterogeneity. (A) Two continuous recordings of WT single channels at −100 mV showing low open probability (top) and high open probability (bottom) currents. (B) A continuous recording of WT single channels at the indicated voltages. (C) Plots of intraburst open probability for every channel burst in a patch of WT and I131C/I138D. The currents above show two bursts at −100 mV with contrasting open probabilities that correspond to the data points indicated by the arrows. Open probability and current amplitude are shown above each burst.
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fig8: Single-channel recordings show significant gating heterogeneity. (A) Two continuous recordings of WT single channels at −100 mV showing low open probability (top) and high open probability (bottom) currents. (B) A continuous recording of WT single channels at the indicated voltages. (C) Plots of intraburst open probability for every channel burst in a patch of WT and I131C/I138D. The currents above show two bursts at −100 mV with contrasting open probabilities that correspond to the data points indicated by the arrows. Open probability and current amplitude are shown above each burst.

Mentions: Single-channel gating kinetics of KirBac1.1 are also complex. Open probability varies significantly from patch to patch. Fig. 8 A shows two WT recordings with contrasting open probabilities. Open probability also tends to be higher at positive voltages than negative voltages, as seen in Fig. 8 B. This may explain why, although WT macroscopic currents show a near linear current–voltage relationship (Fig. 1), single-channel amplitudes exhibit inward rectification (Fig. 7 C). Detailed inspection of these recordings reveals variations in intraburst gating kinetics at negative voltages (Fig. 8 C). Single-channel bursts at −100 mV were individually idealized using a “50% threshold” criterion based on their respective amplitudes determined from all-point histograms (red dashed lines in Fig. 8 C), and the idealized data were used to calculate intraburst open probability. A plot of the intraburst open probability for all bursts in patches of WT and I131C/I138D show at least two gating modes at −100 mV. Generally, most openings have a high P (o) of ∼0.9, but occasionally, openings will switch to a flicker mode where P (o) is ∼0.65. Such gating heterogeneities are uncommon among eukaryotic potassium channels in cellular membranes, but similar behaviors have been reported for KcsA reconstituted in liposomes (Chakrapani et al., 2007b).


KirBac1.1: it's an inward rectifying potassium channel.

Cheng WW, Enkvetchakul D, Nichols CG - J. Gen. Physiol. (2009)

Single-channel recordings show significant gating heterogeneity. (A) Two continuous recordings of WT single channels at −100 mV showing low open probability (top) and high open probability (bottom) currents. (B) A continuous recording of WT single channels at the indicated voltages. (C) Plots of intraburst open probability for every channel burst in a patch of WT and I131C/I138D. The currents above show two bursts at −100 mV with contrasting open probabilities that correspond to the data points indicated by the arrows. Open probability and current amplitude are shown above each burst.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654083&req=5

fig8: Single-channel recordings show significant gating heterogeneity. (A) Two continuous recordings of WT single channels at −100 mV showing low open probability (top) and high open probability (bottom) currents. (B) A continuous recording of WT single channels at the indicated voltages. (C) Plots of intraburst open probability for every channel burst in a patch of WT and I131C/I138D. The currents above show two bursts at −100 mV with contrasting open probabilities that correspond to the data points indicated by the arrows. Open probability and current amplitude are shown above each burst.
Mentions: Single-channel gating kinetics of KirBac1.1 are also complex. Open probability varies significantly from patch to patch. Fig. 8 A shows two WT recordings with contrasting open probabilities. Open probability also tends to be higher at positive voltages than negative voltages, as seen in Fig. 8 B. This may explain why, although WT macroscopic currents show a near linear current–voltage relationship (Fig. 1), single-channel amplitudes exhibit inward rectification (Fig. 7 C). Detailed inspection of these recordings reveals variations in intraburst gating kinetics at negative voltages (Fig. 8 C). Single-channel bursts at −100 mV were individually idealized using a “50% threshold” criterion based on their respective amplitudes determined from all-point histograms (red dashed lines in Fig. 8 C), and the idealized data were used to calculate intraburst open probability. A plot of the intraburst open probability for all bursts in patches of WT and I131C/I138D show at least two gating modes at −100 mV. Generally, most openings have a high P (o) of ∼0.9, but occasionally, openings will switch to a flicker mode where P (o) is ∼0.65. Such gating heterogeneities are uncommon among eukaryotic potassium channels in cellular membranes, but similar behaviors have been reported for KcsA reconstituted in liposomes (Chakrapani et al., 2007b).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

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 (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.

Show MeSH
Related in: MedlinePlus