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Comparative Study
. 2017 Jun 16;292(24):10087-10096.
doi: 10.1074/jbc.M117.785154. Epub 2017 Apr 26.

Conformational changes at cytoplasmic intersubunit interactions control Kir channel gating

Shizhen Wang  1 , William F Borschel  1 , Sarah Heyman  1 , Phillip Hsu  1 , Colin G Nichols  2
Affiliations
Comparative Study

Conformational changes at cytoplasmic intersubunit interactions control Kir channel gating

Shizhen Wang et al. J Biol Chem. .

Abstract

The defining structural feature of inward-rectifier potassium (Kir) channels is the unique Kir cytoplasmic domain. Recently we showed that salt bridges located at the cytoplasmic domain subunit interfaces (CD-Is) of eukaryotic Kir channels control channel gating via stability of a novel inactivated closed state. The cytoplasmic domains of prokaryotic and eukaryotic Kir channels show similar conformational rearrangements to the common gating ligand, phosphatidylinositol bisphosphate (PIP2), although these exhibit opposite coupling to opening and closing transitions. In Kir2.1, mutation of one of these CD-I salt bridge residues (R204A) reduces apparent PIP2 sensitivity of channel activity, and here we show that Ala or Cys substitutions of the functionally equivalent residue (Arg-165) in the prokaryotic Kir channel KirBac1.1 also significantly decrease sensitivity of the channel to PIP2 (by 5-30-fold). To further understand the structural basis of CD-I control of Kir channel gating, we examined the effect of the R165A mutation on PIP2-induced changes in channel function and conformation. Single-channel analyses indicated that the R165A mutation disrupts the characteristic long interburst closed state of reconstituted KirBac1.1 in giant liposomes, resulting in a higher open probability due to more frequent opening bursts. Intramolecular FRET measurements indicate that, relative to wild-type channels, the R165A mutation results in splaying of the cytoplasmic domains away from the central axis and that PIP2 essentially induces opposite motions of the major β-sheet in this channel mutant. We conclude that the removal of stabilizing CD-I salt bridges results in a collapsed state of the Kir domain.

Keywords: fluorescence resonance energy transfer (FRET); gating; membrane transporter reconstitution; phosphatidylinositol; potassium channel.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
The R204A mutation reduced PIP2 sensitivity of Kir2.1. A, sequence alignments of Kir channels using ClustalW; a two-residue gap was introduced to comply with the structural alignments. B, Kir channel cytoplasmic domains of KirBac1.1 (PDB code 2WLL, green), Kir3.2 (3SYP, pink) and Kir2.2 (3SPI, cyan and light blue) were aligned with PyMOL, with Arg-165 of KirBac1.1 and equivalent Arg-215 of Kir3.2, and Arg-204 of Kir2.2 highlighted in the right panel as sticks. C and D, representative macroscopic currents of human Kir2.1 WT (C) and R204A (D) mutants in cell-attached and excised (inside-out) patches in response to a voltage ramp from −100 to +100 mV membrane potential; 4.6 μm μm PIP2 was applied in the bath solution after the patches were excised and the currents reached steady state. E, changes of Kir2.1 WT and R204A channel currents induced by 4.6 μm PIP2. Channel potentiation by PIP2 was calculated using equation % = IPIP2/IExcised−1, where IExcised is the steady-state current at −100 mV in excised mode, and IPIP2 is the steady-state current after PIP2 addition. WT, n = 6; Arg-204, n = 5; *, p < 0.05.
Figure 2.
Figure 2.
The equivalent Arg-165 of KirBac1.1 determined its PIP2 sensitivity. A, MTS reagents with positive charged group reversibly modify the sensitivity of KirBac1.1-R165C to PIP2. Purified KirBac1.1-R165C in buffer containing 20 mm Hepes, 150 mm KCl, and 5 mm DM, pH 7.5, was modified by MTSMT, MTSCM, and MTSEA (protein:MTS-reagents = 1:10) at room temperature for 30 min, then reconstituted into liposomes (POPE:POPG = 3:1) with or without 1% of PIP2; the MTS- modification was removed by 25 mm DTT treatment at room temperature for 30 min. B, mutational analysis of the role of Arg-165 in determining the PIP2 sensitivity of KirBac1.1. Purified KirBac1.1-R165X mutants were reconstituted into liposomes (POPE:POPG = 3:1) containing 0.0, 0.01, 0.1, or 1.0% of PIP2. The relative rubidium uptake at different PIP2 concentrations was determined and normalized as that described under “Experimental Procedures” (mean ± S.E., n = 3 in each case). C, SDS-PAGE analysis of disulfide bond formation between intersubunit R165C and E202C. Monomer, dimer, trimer, and tetramer bands were marked by arrows. KirBac1.1 WT and R165C-E202C mutant (Mu) in the presence and absence of 75 μm diC8-PIP2 were treated with 5 mm H2O2 or tris(2-carboxyethyl)phosphine (TCEP) for 3 h under room temperature. D, densitometry was performed on scanned gel images with ImageJ software, and the tetramer fraction was calculated as density ratio of the tetrameric band to all oligomeric bands in the same lane.
Figure 3.
Figure 3.
Stoichiometric effect of R165A mutation on PIP2 sensitivity of KirBac1.1. A, gel filtration profiles of affinity-purified KirBac1.1 R165A dimeric or tetrameric tandem proteins. The running buffer for all gel filtration was 20 mm Hepes, 150 mm KCl, 5 mm decyl-maltoside, pH 7.5. B, SDS-PAGE of affinity-purified KirBac1.1 mutant proteins with different stoichiometry. C, effect of number and location of R165A mutations within the KirBac1.1 tetramer on PIP2 sensitivity. Ki (the PIP2 concentration at which 50% of KirBac1.1 rubidium uptake was inhibited) values were obtained by fitting with the Hill equation using Solver tool of Microsoft Excel. ΔΔG was calculated using equation ΔΔG = RTln(Ki R165X/Ki WT).
Figure 4.
Figure 4.
R165A enhanced KirBac1.1 activity by reducing the interburst closed time. A and B, representative single channel recordings (above) and single channel current amplitude histogram (below) of purified KirBac1.1 WT and R165A mutant in giant liposomes at +100-mV membrane potential. C, closed lifetime histograms overlaid with the probability density function (thick solid line) of KirBac1.1 WT and R165A mutant, which were fit well with three exponential components (overlaid lines) with the longest component marked by arrows. D, distribution of interburst duration of KirBac1.1 WT and R165A from the entire recording depicted in A and B. E, open probability (Po), mean closed time (MCT) (F), and interburst duration (G) of KirBac1.1 WT and R165A. WT, n = 4 patches; R165A, n = 5 patches in E–G; *, p < 0.05.
Figure 5.
Figure 5.
Correlation analysis revealed significant structural changes in KirBac1.1 due to the R165A mutation. A, relative rubidium uptake of KirBac1.1 cysteine mutants (on the R165A background) labeled with EDANS/DABCYL-plus (E/D) FRET pairs in the presence and absence of 1% of PIP2 (mean ± S.E., n = 3 in each case). Mutants with substantially lower rubidium uptake than WT (>20%, gray) were not analyzed further. B, relative PIP2 inhibition of E/D-labeled KirBac1.1 cysteine mutants on the R165A background. Relative PIP2 inhibition was calculated from A and normalized to WT. All mutants were variably less sensitive than WT (green). C, Cα-Cα distance between adjacent subunits of labeled residues assessed by FRET versus those determined in the KirBac1.1 (PDB code 2WLL) crystal structure. KirBac1.1 cysteine-substituted proteins on the WT or R165A background were purified, labeled, and reconstituted into liposomes (POPE:POPG = 3:1) with or without 1% PIP2, apparent FRET efficiencies were measured, and Cα-Cα distances were calculated using a tetrameric FRET model as that described previously (12). D, schematic illustration of significant structural changes induced by the R165A mutation in KirBac1.1 subunits. The Cα of residues subjected to FRET measurements are highlighted as spheres, with those that move toward the pore axis colored blue and those that move away colored red upon the addition of PIP2 or introduction of R165A background mutation. The major β-sheet uniformly moves away from the central axis (arrow) in the R165A background.
Figure 6.
Figure 6.
The R165A mutation disrupted PIP2-induced secondary structural rearrangements. Shown are PIP2-induced changes of apparent FRET efficiencies (EPIP2 − Econtrol) of KirBac1.1 cysteine mutants on the R165A background in the large β-sheet (A and B) and small β-sheets (D and E). Labeled mutants showing significantly reduced PIP2 inhibition (from Fig. 4) are highlighted by colors. In B and E, Cα of the labeled residue is highlighted by spheres; residues demonstrating increased FRET efficiency (inward motion) in the presence of PIP2 are colored blue, those demonstrating decreased FRET efficiency (outward motion) are colored red; amino acid residues in panels A and D are listed moving from the membrane surface toward the bottom of the channel. PIP2-induced structural rearrangements of KirBac1.1 cysteine mutants on the WT background (12) are shown in panels C and F for comparison, with the same color code as that in panel B and E. * indicates a significance level for p < 0.05, and ** indicates a significance level for p < 0.01.
Figure 7.
Figure 7.
Proposed model of structural and functional role of CD-I salt bridge in Kir channel gating. Shown is a schematic model of KirBac1.1 channel gating. The WT CD-I salt bridge (circled) stabilizes the closed state, whereas in the R165A mutant channel, the CD-I is destabilized, and Po is increased. PIP2 binds to the “tight” closed state cytoplasmic domain conformation, thereby reducing apparent PIP2-sensitivity in the R165A mutant channel.
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