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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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The I131C/I138D mutant is more sensitive to spermine inhibition than WT. (A) Structure of two opposing subunits of KirBac1.1 highlighting the residues I131 (blue) and I138 (red) that were mutated to cysteine and asparate, respectively. R49 is also highlighted in yellow. Mutation of this residue to a cysteine renders an inactive channel that can be rescued by MTSET modification. (B) Plot of relative 86Rb+ uptake of WT and I131C/I138D in liposomes at different concentrations of externally applied spermine, normalized to uptake without spermine (n = 9 ± SEM). The I131C/I138D data are fit with the sum of two Hill functions (K1/2 = 1 µM, H = 1; K1/2 = 15 mM, H = 0.8), and the WT data with one (K1/2 = 0.8 mM, H = 0.6).
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fig3: The I131C/I138D mutant is more sensitive to spermine inhibition than WT. (A) Structure of two opposing subunits of KirBac1.1 highlighting the residues I131 (blue) and I138 (red) that were mutated to cysteine and asparate, respectively. R49 is also highlighted in yellow. Mutation of this residue to a cysteine renders an inactive channel that can be rescued by MTSET modification. (B) Plot of relative 86Rb+ uptake of WT and I131C/I138D in liposomes at different concentrations of externally applied spermine, normalized to uptake without spermine (n = 9 ± SEM). The I131C/I138D data are fit with the sum of two Hill functions (K1/2 = 1 µM, H = 1; K1/2 = 15 mM, H = 0.8), and the WT data with one (K1/2 = 0.8 mM, H = 0.6).

Mentions: WT KirBac1.1 behaves as a weak inward rectifier and is insensitive to block by spermine in both patch clamp and 86Rb+ flux assays (Figs. 3 B and 4 A). We hypothesized that mutation of I138, which is equivalent to the rectification controller residue in eukaryotic Kir channels, to an aspartate would confer sensitivity to polyamines just as in Kir1.1 (N171D) and Kir6.2 (N160D; Fig. 3 A, red). However, multiple preparations of I138D mutant KirBac1.1 were inactive when tested by the 86Rb+ flux assay (not depicted). We have found that mutation of residue 131 (located in the upper region of TM2, adjacent to the base of the pore helix and near the selectivity filter; Fig. 3 A, blue) from isoleucine to cysteine stabilizes the channel tetramer in SDS-PAGE (Wang et al., 2008). On the I131C background, the I138D mutant generated functional channels assessed by 86Rb+ flux (Fig. 3 B). 86Rb+ flux assays of WT and I131C/I138D in different concentrations of spermine illustrate that the double mutant, I131C/I138D, is strongly inhibited by spermine, and the concentration dependence of inhibition shows two distinct components. The I131C/I138D data can be fit with the sum of two Hill equations (K1/2 = 1 µM and 15 mM), and the WT data with a single Hill equation (K1/2 = 0.8 mM). The fact that only 50% of 86Rb+ uptake is inhibited with high affinity is consistent with the generation of high affinity block only to internal/cytoplasmic spermine in I131C/I138D, and with channels being bi-directionally oriented (see Discussion).


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

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

The I131C/I138D mutant is more sensitive to spermine inhibition than WT. (A) Structure of two opposing subunits of KirBac1.1 highlighting the residues I131 (blue) and I138 (red) that were mutated to cysteine and asparate, respectively. R49 is also highlighted in yellow. Mutation of this residue to a cysteine renders an inactive channel that can be rescued by MTSET modification. (B) Plot of relative 86Rb+ uptake of WT and I131C/I138D in liposomes at different concentrations of externally applied spermine, normalized to uptake without spermine (n = 9 ± SEM). The I131C/I138D data are fit with the sum of two Hill functions (K1/2 = 1 µM, H = 1; K1/2 = 15 mM, H = 0.8), and the WT data with one (K1/2 = 0.8 mM, H = 0.6).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: The I131C/I138D mutant is more sensitive to spermine inhibition than WT. (A) Structure of two opposing subunits of KirBac1.1 highlighting the residues I131 (blue) and I138 (red) that were mutated to cysteine and asparate, respectively. R49 is also highlighted in yellow. Mutation of this residue to a cysteine renders an inactive channel that can be rescued by MTSET modification. (B) Plot of relative 86Rb+ uptake of WT and I131C/I138D in liposomes at different concentrations of externally applied spermine, normalized to uptake without spermine (n = 9 ± SEM). The I131C/I138D data are fit with the sum of two Hill functions (K1/2 = 1 µM, H = 1; K1/2 = 15 mM, H = 0.8), and the WT data with one (K1/2 = 0.8 mM, H = 0.6).
Mentions: WT KirBac1.1 behaves as a weak inward rectifier and is insensitive to block by spermine in both patch clamp and 86Rb+ flux assays (Figs. 3 B and 4 A). We hypothesized that mutation of I138, which is equivalent to the rectification controller residue in eukaryotic Kir channels, to an aspartate would confer sensitivity to polyamines just as in Kir1.1 (N171D) and Kir6.2 (N160D; Fig. 3 A, red). However, multiple preparations of I138D mutant KirBac1.1 were inactive when tested by the 86Rb+ flux assay (not depicted). We have found that mutation of residue 131 (located in the upper region of TM2, adjacent to the base of the pore helix and near the selectivity filter; Fig. 3 A, blue) from isoleucine to cysteine stabilizes the channel tetramer in SDS-PAGE (Wang et al., 2008). On the I131C background, the I138D mutant generated functional channels assessed by 86Rb+ flux (Fig. 3 B). 86Rb+ flux assays of WT and I131C/I138D in different concentrations of spermine illustrate that the double mutant, I131C/I138D, is strongly inhibited by spermine, and the concentration dependence of inhibition shows two distinct components. The I131C/I138D data can be fit with the sum of two Hill equations (K1/2 = 1 µM and 15 mM), and the WT data with a single Hill equation (K1/2 = 0.8 mM). The fact that only 50% of 86Rb+ uptake is inhibited with high affinity is consistent with the generation of high affinity block only to internal/cytoplasmic spermine in I131C/I138D, and with channels being bi-directionally oriented (see Discussion).

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