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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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Continuous recording of R49C held at +100 and −100 mV. 0.1 mM MTSET solution was applied as shown by the arrow, and solution changes were made at the indicated bars. Below are all-points histograms of the recording above with representative traces at +100 and −100 mV. The red dashed lines indicate current amplitudes that correspond with peaks in the histograms.
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fig9: Continuous recording of R49C held at +100 and −100 mV. 0.1 mM MTSET solution was applied as shown by the arrow, and solution changes were made at the indicated bars. Below are all-points histograms of the recording above with representative traces at +100 and −100 mV. The red dashed lines indicate current amplitudes that correspond with peaks in the histograms.

Mentions: In a prior study, using the 86Rb+ flux assay, we showed that cysteine mutants of KirBac1.1 in the slide helix can be activated by modification with MTS reagents. In particular, the mutations R49C or K57C render the channel inactive, but can be rescued by MTSEA or MTSET modification (Enkvetchakul et al., 2007). KirBac1.1 channels reconstituted in giant liposomes are not necessarily oriented in the same direction as eukaryotic channels expressed in cell membranes. Because R49 is located in the slide helix (Fig. 3 A, yellow), application of MTSET in the bath can only activate those channels in which the “cytoplasmic” end is also facing the bath. Fig. 9 shows a continuous recording of R49C single-channel activity after activation by MTSET. The currents behave exactly as WT: they are inhibited by 1 mM Ba2+ but insensitive to 0.1 mM spermine. Amplitude histograms as well as the general appearance of channel gating at +100 mV compared with −100 mV are similar to WT and I131C/I138D (Figs. 7, 8, and 9). The fact that WT and I131C/I138D exhibit similar single-channel amplitudes and gating behavior as activated R49C is consistent with most of the reconstituted channels being oriented with their cytoplasmic end facing the bath in an inside-out patch.


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

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

Continuous recording of R49C held at +100 and −100 mV. 0.1 mM MTSET solution was applied as shown by the arrow, and solution changes were made at the indicated bars. Below are all-points histograms of the recording above with representative traces at +100 and −100 mV. The red dashed lines indicate current amplitudes that correspond with peaks in the histograms.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig9: Continuous recording of R49C held at +100 and −100 mV. 0.1 mM MTSET solution was applied as shown by the arrow, and solution changes were made at the indicated bars. Below are all-points histograms of the recording above with representative traces at +100 and −100 mV. The red dashed lines indicate current amplitudes that correspond with peaks in the histograms.
Mentions: In a prior study, using the 86Rb+ flux assay, we showed that cysteine mutants of KirBac1.1 in the slide helix can be activated by modification with MTS reagents. In particular, the mutations R49C or K57C render the channel inactive, but can be rescued by MTSEA or MTSET modification (Enkvetchakul et al., 2007). KirBac1.1 channels reconstituted in giant liposomes are not necessarily oriented in the same direction as eukaryotic channels expressed in cell membranes. Because R49 is located in the slide helix (Fig. 3 A, yellow), application of MTSET in the bath can only activate those channels in which the “cytoplasmic” end is also facing the bath. Fig. 9 shows a continuous recording of R49C single-channel activity after activation by MTSET. The currents behave exactly as WT: they are inhibited by 1 mM Ba2+ but insensitive to 0.1 mM spermine. Amplitude histograms as well as the general appearance of channel gating at +100 mV compared with −100 mV are similar to WT and I131C/I138D (Figs. 7, 8, and 9). The fact that WT and I131C/I138D exhibit similar single-channel amplitudes and gating behavior as activated R49C is consistent with most of the reconstituted channels being oriented with their cytoplasmic end facing the bath in an inside-out patch.

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