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PLoS One
2013 Jan 01;811:e79844. doi: 10.1371/journal.pone.0079844.
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Conformational changes underlying pore dilation in the cytoplasmic domain of mammalian inward rectifier K+ channels.
Inanobe A
,
Nakagawa A
,
Kurachi Y
.
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The cytoplasmic domain of inward rectifier K(+) (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu236 and Met313, of the G protein-gated Kir channel subunit Kir3.2. In the closed state, these pore-facing residues are present on adjacent βD and βH strands, respectively. We mutated both residues, expressed them with the m2-muscarinic receptor in Xenopus oocytes, and measured the acetylcholine-dependent K(+) currents. The dose-response curves of the Glu236 mutants tended to be shifted to the right. In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants. The introduction of arginine at position 236 conferred constitutive activity and caused a leftward shift in the conductance-voltage relationship. The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the βH and βI strands of the adjacent subunit. Because the βH strand forms a β sheet with the βI and βD strands, the immobilization of the pore-forming β sheet appears to confer unique properties to the mutant. These results suggest that the G protein association triggers pore dilation at the cytoplasmic domain in functional channels, and the pore-constituting structural elements contribute differently to these conformational changes.
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Figure 2. Effects of mutations at Glu236 and Met313 on m2R-dependent activation.A. Typical current traces of Kir3.2 wild-type (WT) and mutant channels recorded in the presence of various concentrations of ACh. The current responses were elicited by a test pulse at −120 mV for 0.6 s followed by a voltage step to +40 mV for 0.6 s at an interval of 0.1 s at −20 mV. This sequence was repeated every 3 s. The current in the presence of 3 mM Ba2+ was subtracted from each trace. The arrowheads indicate the zero current levels.B, C. Current response of Kir3.2 WT and mutant channels. ACh-induced K+ currents recorded at the end of the test pulse to −120 mV were normalized to that measured in the presence of 10 μM ACh. Error bars indicate the standard error of the mean. The current responses of the WT and mutants were fit using the Hill equation as described in the Materials and Methods section.
Figure 3. Effects of mutations at position 313 of Kir3.2 on basal activity.Comparison of the ratios of the basal current of Kir3.2 WT and mutant channels.The current amplitudes of Kir3.2 WT and mutant channels in the absence of ACh (Ibasal) were divided by those in the presence of 10 μM ACh (Itotal) in every oocyte. The Ibasal/Itotal ratio was plotted against the amplitude of Itotal for the WT and each mutant. The plot shows a linear relationship when the Itotal is less than 10 μA.
Figure 4. Effects of mutations at position 313 of Kir3.2 on single-channel currents.A. Single-channel recording of the indicated channel at −100 mV. The patch recording in the cell-attached configuration was obtained from HEK293T cells expressing Kir3.2 WT, M313G, and M313T. The holding potential was −100 mV. Open dwell time histograms for each channel are shown at the right side of the trace and are fit with a single exponential function. The single-channel current amplitudes of the WT and M313G mutant are plotted against the membrane potentials.B. Effects of substitutions of threonine 301 in Kir2.1.Met301 in Kir2.1 is equivalent to Met313 in Kir3.2. The substitution of threonine at Met301 in Kir2.1 resulted in a spiky opening with variable conductance. Arrowheads indicate the zero current level.
Figure 5. Inside-out patch recordings of Kir3.2 WT and mutant channels.Inside-out membrane patches were obtained from HEK293T cells expressing either Kir3.2 WT (A), M313G (B) or M313T mutant (C) channels. Experiments were conducted in symmetric 135 mM K+ solutions with a holding potential of -100 mV. Perfusion of ATP was for the generation of PIP2 at the inner leaflet of the excised patch membranes. The protocol used for perfusion of substances to the intracellular side of the patch membrane is indicated by bars above the current traces.
Figure 6. Introduction of reverse charge at position 236 of Kir3.2.A. Current traces of the Kir3.2 E236R mutant. Typical current traces of the E236R mutant recorded in the presence of various concentrations of ACh are shown. The Ba2+-insensitive current component was subtracted. Arrowheads indicate zero current levels.B. Whole-cell current amplitudes of the E236R mutant. The K+ currents recorded at the end of the test pulse to −120 mV show that the mutant is insensitive to m2R-stimulation. The arrow indicates the time at which Ba2+ was loaded into the bath solution.C. Whole-cell currents from oocytes expressing Kir3.2 WT and E236R mutant channels. Macroscopic currents were induced by voltage steps (1.2 s) from the holding potential of −20 mV to potentials from −140 to +80 mV in 20-mV increments. The Ba2+-sensitive currents of the WT and E236R channels in the presence of 10 μM ACh are shown. D. Current-voltage relationship. The current amplitudes at the end of the test pulse were normalized to that recorded at -100 mV. E. Conductance-voltage relationship. The voltage dependence of channel activation was determined from the relationship of chord conductance to voltage according to the following Boltzmann equation.g/gmax=gmin+(1−gmin)/(1+exp[{V−V1/2}/k])where V1/2 is the half-maximal activation voltage and k is the slope factor: WT: V1/2 = −34 ± 4 mV, k = 27 ± 3 mV (n = 10); E236R: V1/2 = −78 ± 1 mV, k = 28 ± 1 mV (n = 11). Error bars indicate the standard error of the mean. All error bars are smaller than the symbols used.F. Single-channel recording of E236R. Channel currents were recorded from HEK293T cells in the cell-attached patch-clamp configuration. The arrowhead to the left of the trace represents the zero current level.
Figure 7. Crystal structure of the Kir3.2 E236R mutant.A. Comparison of the crystal structures of the cytoplasmic domains (CPDs) of WT and E236R channels. The CPD of the E236R mutant (blue) behaves as a tetramer and has a structure similar to that of the WT in a closed conformation (gray). The βC and βD strands (CD loop; yellow) and HI loop (orange) are denoted by circles in 1 subunit and traced with lines in the next subunit.B. Enlarged view of the subunit interface at position 236. An arginine introduced at site 236 of one subunit (blue) forms hydrogen bonds with the carbonyl oxygen atoms of Gly312 on the βH strand and Cys321 on the βI strand of the adjacent subunit (gray). The residues crucial for the interaction are shown as sticks.C. The β-bulge in the βD strand. The β sheet observed from the pore is shown with ribbons and sticks. The side chains of Glu236 and Met313 are shown with dots. The β-type hydrogen bonds are disconnected between Glu236 and Gly237 on the βD strand and Gly312 on βH strand. The dashed line indicates the hydrogen bond between the main chain atoms.
Figure 1. Molecular architecture of Kir3.2.
A. Domain topology of Kir3.2. The Kir channel comprises the transmembrane domain (TMD) and cytoplasmic domain (CPD). The ion permeation pathway is located at the center of the tetrameric assembly. Glu236 and Met313 are present on adjacent βD and βH strands, respectively.
B, C. Enlarged side (B) and top (C) views of the region of the CPD of Kir3.2 near the TMD. The ribbon trace represents the Cα backbone of the Kir3.2 model, and Glu236 and Met313 are shown as sticks and spheres. For clarity, the front and rear subunits in the assembly are omitted from the side view (B).
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