Fig. 3. α-MSH and AgRP couple MC4R to Kir7.1 in HEK293 cells.
a–c, Current responses of transfected HEK293 cells to voltage steps (a) and ramps (b) in control, 300nM α-MSH, and washout. Bath application of α-MSH significantly reduced the amplitude (c) and slope of responses to voltage steps and ramps in a reversible manner (bar graph indicates mean +/−SEM; *p<0.05, ***p<0.01, n=6, paired t test). d–g, α-MSH reduces flux (IC50 near 10−7.5 M, d & e) and AgRP increases flux (EC50 near 10−8.6 M, h & i) through Kir7.1 channels in a concentration–dependent manner. Concentration-response curves in e & g are plotted using the maxima from data in d & f. h–k, Specificity of melanocortin receptor-Kir subunit coupling. h, α-MSH -induced decrease in Tl+ flux in MC4R and MC1R expressing cells containing Kir7.1, but not in cells co-expressing Kir7.1 and the MC3R, or expressing Kir7.1 alone. i, α-MSH -induced decrease in Tl+ flux via the MC4R is observed in cells transfected with Kir7.1, more weakly in cells expressing Kir4.1, and is not in cells expressing Kir2.1 or Kir2.3. j, VU573 (10μM) blocks Tl+ flux in MC4R+Kir7.1 transfected cells. k, VU573 does not block MC4R-Kir4.1 mediated Tl+ flux in the transfected cell assay. l–m, Pre-incubation with 100nM α-MSH of MC4R-glo expressing HEK293 cells directly depresses a cAMP test response to a second exposure to 100nM α-MSH (l). Pre-incubation MC4R-Kir7.1 HEK293 cells with 100nM α-MSH directly increases the amplitude of the response of Kir7.1 to a second 100nM α-MSH exposure (m). n, Kinetics of the normalized maximal cAMP (black) and Kir7.1 (red) response to a single dose of 100nM α-MSH, calculated from Figs. l–m. In all Tl+ flux and cAMP accumulation assays, colored traces indicate mean and bars (black) indicate SEM.