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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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KirBac1.1 voltage-clamped currents are potassium selective and blocked by barium. Inside-out patches were excised from giant liposomes reconstituted with (A) no protein or (B) WT KirBac1.1. (A) Representative currents from an inside-out patch from giant liposomes without KirBac1.1 protein. The pipette was filled with 158 mM K+ and the bath with the indicated solutions. The voltage was held at −50 mV and then stepped from −100 to +100 mV at 10-mV increments. (B) Representative currents from giant liposomes reconstituted with KirBac1.1 as in A. (C) Plot of normalized current versus voltage for WT KirBac1.1 in the solutions shown in B (n = 6 ± SEM). (D) Relative conductance versus voltage plot of KirBac1.1 block in 1 mM Ba2+ (n = 6 ± SEM). Fitting with a Boltzmann function gives the following parameter values: V1/2 = 25 mV, δz = 1.5, residual GREL = 0.08, scaling factor = 0.9.
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fig1: KirBac1.1 voltage-clamped currents are potassium selective and blocked by barium. Inside-out patches were excised from giant liposomes reconstituted with (A) no protein or (B) WT KirBac1.1. (A) Representative currents from an inside-out patch from giant liposomes without KirBac1.1 protein. The pipette was filled with 158 mM K+ and the bath with the indicated solutions. The voltage was held at −50 mV and then stepped from −100 to +100 mV at 10-mV increments. (B) Representative currents from giant liposomes reconstituted with KirBac1.1 as in A. (C) Plot of normalized current versus voltage for WT KirBac1.1 in the solutions shown in B (n = 6 ± SEM). (D) Relative conductance versus voltage plot of KirBac1.1 block in 1 mM Ba2+ (n = 6 ± SEM). Fitting with a Boltzmann function gives the following parameter values: V1/2 = 25 mV, δz = 1.5, residual GREL = 0.08, scaling factor = 0.9.

Mentions: Excised patch clamp experiments were performed using giant liposomes reconstituted with or without KirBac1.1 in symmetrical, 158-mM KCl except as indicated (Fig. 1 A). Patches from liposomes without protein yielded minimal currents. In contrast, recordings from liposomes reconstituted with KirBac1.1 show significant current with a near linear voltage–current relationship in symmetrical K+ conditions. When bath K+ is lowered to 28 mM, there is a +31 ± 1 mV (n = 6; SEM) shift in the reversal potential. The largest shift observed was +34 mV, indicating a minimum K+/Na+ permeability ratio of 10:1. Current at positive voltages is blocked by 1 mM Ba2+ (Fig. 1 B), with a V1/2 of 25 mV and a zδ of 1.5 (Fig. 1 C). The data indicate that these currents are due to functional potassium-selective channels.


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

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

KirBac1.1 voltage-clamped currents are potassium selective and blocked by barium. Inside-out patches were excised from giant liposomes reconstituted with (A) no protein or (B) WT KirBac1.1. (A) Representative currents from an inside-out patch from giant liposomes without KirBac1.1 protein. The pipette was filled with 158 mM K+ and the bath with the indicated solutions. The voltage was held at −50 mV and then stepped from −100 to +100 mV at 10-mV increments. (B) Representative currents from giant liposomes reconstituted with KirBac1.1 as in A. (C) Plot of normalized current versus voltage for WT KirBac1.1 in the solutions shown in B (n = 6 ± SEM). (D) Relative conductance versus voltage plot of KirBac1.1 block in 1 mM Ba2+ (n = 6 ± SEM). Fitting with a Boltzmann function gives the following parameter values: V1/2 = 25 mV, δz = 1.5, residual GREL = 0.08, scaling factor = 0.9.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: KirBac1.1 voltage-clamped currents are potassium selective and blocked by barium. Inside-out patches were excised from giant liposomes reconstituted with (A) no protein or (B) WT KirBac1.1. (A) Representative currents from an inside-out patch from giant liposomes without KirBac1.1 protein. The pipette was filled with 158 mM K+ and the bath with the indicated solutions. The voltage was held at −50 mV and then stepped from −100 to +100 mV at 10-mV increments. (B) Representative currents from giant liposomes reconstituted with KirBac1.1 as in A. (C) Plot of normalized current versus voltage for WT KirBac1.1 in the solutions shown in B (n = 6 ± SEM). (D) Relative conductance versus voltage plot of KirBac1.1 block in 1 mM Ba2+ (n = 6 ± SEM). Fitting with a Boltzmann function gives the following parameter values: V1/2 = 25 mV, δz = 1.5, residual GREL = 0.08, scaling factor = 0.9.
Mentions: Excised patch clamp experiments were performed using giant liposomes reconstituted with or without KirBac1.1 in symmetrical, 158-mM KCl except as indicated (Fig. 1 A). Patches from liposomes without protein yielded minimal currents. In contrast, recordings from liposomes reconstituted with KirBac1.1 show significant current with a near linear voltage–current relationship in symmetrical K+ conditions. When bath K+ is lowered to 28 mM, there is a +31 ± 1 mV (n = 6; SEM) shift in the reversal potential. The largest shift observed was +34 mV, indicating a minimum K+/Na+ permeability ratio of 10:1. Current at positive voltages is blocked by 1 mM Ba2+ (Fig. 1 B), with a V1/2 of 25 mV and a zδ of 1.5 (Fig. 1 C). The data indicate that these currents are due to functional potassium-selective channels.

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