Structural and functional studies of inward rectifier Kir7.1 and its regulation by the Melanocortin-4 receptor

Figures

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  Fig. 1. Structures of apo and ML418 bound R162Q mutant of human Kir7.1. A Overlay of Superdex 200 gel filtration profiles of apo and ML418 bound R162Q mutant Kir7.1. B Overlay of Superdex 200 gel filtration profile of apo wild-type (WT) Kir7.1 compared to ML418 bound to the R162Q mutant. C Cartoon representation of cryo-EM structure obtained of apo R162Q mutant (gray, residues 14-325 (excluding residues 31-36), gray) and R162Q mutant bound with ML418 (blue, residues 14-325 (excluding residues 30-39, 161-165) blue). Downward shift of the cytosolic domain (CTD) relative to the transmembrane domain (TMD) is demarcated. D-F Superposition of apo (gray) and ML418 (blue) bound R162Q mutant structures: side view of TMD and CTD. D extracellular view of TMD (E) and cytoplasmic view of CTD (F). Rotation of the cytoplasmic domains in the presence of ML418 is indicated. G-H Backbone trace of selectivity filter residues showing distances between backbone carbonyls in apo R162Q with potassium ions displayed as purple spheres (G). I ML418 bound R162Q with an unoccupied selectivity filter (as in H). Pore radius calculated between van der Waals surfaces by the program HOLE and plotted as a function of the…

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  Fig. 2. Location of ML418 binding site. A Cartoon representation of ML418 bound R162Q mutant of Kir7.1 displaying density observed in the central region of pore cavity. B View across the transmembrane region showing the ML418 binding pose and nearby interacting residues beneath the selectivity filter. C Simplified view of ML418 surrounded by residues within interaction distance. D Chemical structure representation of ML418. E ML418 fitted to density (displayed as mesh) observed in cross-section of central pore region. F Density displayed as mesh surrounding ML418 and Glu149 in side view.

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  Fig. 3. Inhibition of Kir7.1 decreases food intake and promotes weight loss independent of adiposity. A Food intake of wild-type C57BL/6 J mice over a 24-h period treated with 13% DMSO in saline (vehicle control plotted as 0 mg/kg) or ML418 (5 mg/kg or 7.5 mg/kg); 0 mg/kg n = 13; ML418 5 mg/kg n = 6; ML418 7.5 mg/kg n = 7, one-way ANOVA, F (2, 23) = 21.87, p = 0.0001. B 24-h change in body weight in wild-type mice treated with 13% DMSO in saline (vehicle control plotted as 0 mg/kg) or ML418 (5 mg/kg or 7.5 mg/kg); 0 mg/kg n = 13; ML418 5 mg/kg n = 6; ML418 7.5 mg/kg n = 7, one-way ANOVA, F (2, 22) = 9.988, p = 0.008. C Water intake of wild-type mice over 24-h period treated with vehicle (13% DMSO in saline) or ML418 (7.5 mg/kg), n = 5, Student’s t-test (2-tailed), t(8) = 1.361, p = 0.2106. D Food intake in wild-type mice treated with A-4166 (5 mg/kg; A-4166 results are mostly obscured by vehicle values) or vehicle (13% DMSO in saline), n = 5, two-way ANOVA, F (4, 40) = 0.06315, p = 0.9924, multiple comparisons corrected using the Tukey–Kramer method. E Acute food intake over 24-h in wild-type mice centrally treated with vehicle (1μL DMSO) or ML418 (500picomoles, 1μL), n = 9, two-way…

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  Fig. 4. ML418 activates and depolarizes PVH MC4R neurons and blocks further depolarization by a-MSH. A–C Representative voltage traces recorded in current clamp mode (A) demonstrating the effects of α-MSH (500 nM) on the membrane potential (B, from −75 + /−2.8 mV to −65.4 + /−2.8 mV, p = 1.1E-07) and action potential firing frequency (C, from 0.3 + /− 0.2 Hz to 1.9 + /− 0.6 Hz, p = 0.0079) of PVH MC4R neurons (two tailed paired t test, n = 10 cells). D–F Representative voltage trace (D) demonstrating the effects of bath application of ML418 10 µM compared to baseline vehicle on the membrane potential (E, from −68.9 + /− 1.5 mV to −58.7 + /− 2.3 mV, p = 0.004) and action potential firing frequency (F, from 0.05 + /− 0.02 Hz to 0.25 + /− 0.07 Hz, p = 0.011) of PVH MC4R neurons (two tailed paired t test, n = 9 cells). G, H Representative voltage (upper panel) and current (lower panel) traces (G) demonstrating the effects of ML418 10 µM treatment on the number of action potential firings (H, from 2.7 to 4.3 in 100 ms, p = 0.0002) in response to consistent current pulses in baseline vehicle and in ML418 in PVH MC4R neurons (two tailed paired t test, n = 8 cells). I–K A representative voltage…

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  Fig. 5. ML418 activates PVH MC4R neurons and inhibits food intake, and deletion of Kcnj13 from Mc4r-expressing cells blunts ML418 action. A Representative image of coronal sections of the paraventricular hypothalamus (PVH) from MC4R-GFP mice, systemically stimulated with vehicle (DMSO) or ML418. B Quantitation of cells expressing c-Fos in the PVH after animals were treated with vehicle or ML418, n = 5, Student’s t-test (2-tailed), t(8) = 13.26, p = 0.0001. C Percent of c-Fos-positive cells are MC4R cells, Student’s t-test (2-tailed), n = 5, t(8) = 6.6109, p = 0.0002. D 24 hr food intake of Intra-PVH cannulated wild-type mice receiving central injection of vehicle (0.5μL/side, Ringer’s solution, n = 7), or setmelanotide (250pmoles/0.5μL/side, n = 9), Two-way ANOVA: F(5,84) = 2.258, p = 0.0559; multiple comparisons corrected using the Tukey–Kramer method. E 24-h body weight change of the same mice (n = 7 (vehicle) or n = 9 (setmelanotide)), Student’s t-test (2-tailed), t(15) = 2.908, p = 0.0108. F Food intake of Intra-PVH cannulated wild-type mice receiving central injection of vehicle (0.5μL/side, Ringer’s solution) or ML418 (250pmoles/0.5μL/side), n = 8 Two-way ANOVA: F (5, 84) = 3.09, p…

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  Fig. 6. Creation of a linkered MC4r-Kir7.1 protein that retains receptor-mediated channel regulation. A Structure of linkered MC4R-Kir7.1 constructs with no linker or linkers of indicated sequence (L1 through L3). B Intracellular cAMP production concentration-response curves in cells transfected with the GScAMP22f luciferase cAMP genetic sensor and the indicated MC4R or Kir7.1 tethered constructs. The data represent the mean ± S.E.M. from fifteen replicates, with the exception of the MC4R curves based on 12 replicates, distributed in three assay plates. The data were fitted to a four-parameter sigmoid model. C pEC50 values and (D) maximum effect (EMAX) relative to the control MC4R construct transfection obtained from the concentration-response curves shown in (B). The data in (D) represent the mean ± S.E.M of EMAX values determined from each of the three assay plates, Significance (D, E) was determined by one-way ANOVA and the Dunnett post-test. *= p < 0.05, ** = p < 0.01, *** = p < 0.001,**** = p < 0.0001. E Representative current traces of the MC4R-Kir7.1 tethered constructs are shown, evoked from a holding potential of −60 mV to a step pulse of −140 applied for 200 ms. F Concentration…