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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 openings of KirBac1.1 show multiple conductance levels. (A) The currents shown are representative single-channel openings at −100 mV at all amplitude levels from a patch of WT and I131C/I138D. (B) All-points histograms of all channel openings from each recording. (C) A compilation of all-points histograms of channel openings from WT (n = 4; black) and I131C/I138D (n = 3; blue) recordings. The baseline (zero-channel current) peak is included for one histogram only (gray), and each histogram is scaled, for the sake of comparison, by dividing the bin values by the total number of points in each respective histogram.
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fig6: Single-channel openings of KirBac1.1 show multiple conductance levels. (A) The currents shown are representative single-channel openings at −100 mV at all amplitude levels from a patch of WT and I131C/I138D. (B) All-points histograms of all channel openings from each recording. (C) A compilation of all-points histograms of channel openings from WT (n = 4; black) and I131C/I138D (n = 3; blue) recordings. The baseline (zero-channel current) peak is included for one histogram only (gray), and each histogram is scaled, for the sake of comparison, by dividing the bin values by the total number of points in each respective histogram.

Mentions: Single-channel currents of KirBac1.1 also show complex properties, most obviously multiple conductance states. Fig. 6 A shows representative channel openings from two individual patches of WT and I131C/I138D. Both exhibit at least five conductance states. All-points histograms of all channel openings in these two recordings show that smaller conductance states predominate (Fig. 6 B). During bursts of smaller amplitude openings, short transitions to larger conductance states are typically present. The largest single-channel amplitude observed for WT had a conductance of ∼46 pS (chord conductance at −100 mV), and for I131C/I138D, ∼56 pS. However, all single-channel recordings primarily showed two smaller conductance states: the most prevalent at ∼15 pS, and a larger opening at ∼32 pS, measured at −100 mV (Fig. 6 C). These histograms only include channel openings; the area of the baseline peak (Fig. 6 C shows only one in gray) gives no indication of open probability. Fig. 7 A shows all-points histograms of all single-channel openings at +100 and −100 mV from a WT patch, with representative traces from the recording shown above. The red dotted lines in the traces indicate the current levels that correspond with the peaks in the histograms. The two predominant conductance peaks shift to a lower amplitude at +100 mV compared with −100 mV, and stepping from −100 to +100 mV during a single-channel burst shows that the ∼32-pS conductance state at −100 mV corresponds to a conductance of ∼21 pS at +100 mV (Fig. 7 B). All-points histograms were used to determine the major single-channel amplitudes over a range of voltages. Plots of single-channel amplitude versus voltage show that the two major conductance states exhibit a mild intrinsic inward rectification in WT and I131C/I138D (Fig. 7 C; n = 4).


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

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

Single-channel openings of KirBac1.1 show multiple conductance levels. (A) The currents shown are representative single-channel openings at −100 mV at all amplitude levels from a patch of WT and I131C/I138D. (B) All-points histograms of all channel openings from each recording. (C) A compilation of all-points histograms of channel openings from WT (n = 4; black) and I131C/I138D (n = 3; blue) recordings. The baseline (zero-channel current) peak is included for one histogram only (gray), and each histogram is scaled, for the sake of comparison, by dividing the bin values by the total number of points in each respective histogram.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Single-channel openings of KirBac1.1 show multiple conductance levels. (A) The currents shown are representative single-channel openings at −100 mV at all amplitude levels from a patch of WT and I131C/I138D. (B) All-points histograms of all channel openings from each recording. (C) A compilation of all-points histograms of channel openings from WT (n = 4; black) and I131C/I138D (n = 3; blue) recordings. The baseline (zero-channel current) peak is included for one histogram only (gray), and each histogram is scaled, for the sake of comparison, by dividing the bin values by the total number of points in each respective histogram.
Mentions: Single-channel currents of KirBac1.1 also show complex properties, most obviously multiple conductance states. Fig. 6 A shows representative channel openings from two individual patches of WT and I131C/I138D. Both exhibit at least five conductance states. All-points histograms of all channel openings in these two recordings show that smaller conductance states predominate (Fig. 6 B). During bursts of smaller amplitude openings, short transitions to larger conductance states are typically present. The largest single-channel amplitude observed for WT had a conductance of ∼46 pS (chord conductance at −100 mV), and for I131C/I138D, ∼56 pS. However, all single-channel recordings primarily showed two smaller conductance states: the most prevalent at ∼15 pS, and a larger opening at ∼32 pS, measured at −100 mV (Fig. 6 C). These histograms only include channel openings; the area of the baseline peak (Fig. 6 C shows only one in gray) gives no indication of open probability. Fig. 7 A shows all-points histograms of all single-channel openings at +100 and −100 mV from a WT patch, with representative traces from the recording shown above. The red dotted lines in the traces indicate the current levels that correspond with the peaks in the histograms. The two predominant conductance peaks shift to a lower amplitude at +100 mV compared with −100 mV, and stepping from −100 to +100 mV during a single-channel burst shows that the ∼32-pS conductance state at −100 mV corresponds to a conductance of ∼21 pS at +100 mV (Fig. 7 B). All-points histograms were used to determine the major single-channel amplitudes over a range of voltages. Plots of single-channel amplitude versus voltage show that the two major conductance states exhibit a mild intrinsic inward rectification in WT and I131C/I138D (Fig. 7 C; n = 4).

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