Fig. 2. α-MSH depolarizes and AgRP hyperpolarizes hypothalamic PVN MC4R neurons by regulating Kir7.1.

a–c; g, Current-voltage (I–V) analysis of MC4R PVN neurons indicates that 250nM α-MSH generates depolarizing current by closure of inward rectifying K⁺ channels. d–g, I–V analysis of PVN MC4R neurons indicates that 50nM AgRP generates hyperpolarizing current by opening of inward rectifying K⁺ channels. Current responses of voltage clamped PVN neurons to voltage ramps (a&d) were used to generate the I–V relationship of the α-MSH and AgRP-induced response in the presence of 20 mM external K⁺. The resultant I–V relationships (b&e) indicate that application of α-MSH and AgRP, respectively, decrease and increase the cell membrane conductance which rectifies inwardly at membrane potentials negative to its reversal of polarity that are near the estimated reversal potential of K⁺ (α-MSH, n=14, b–c; g, AgRP, n=6, e–g). h–j, Depletion of intracellular Mg²⁺ significantly increases K+ efflux through MC4R regulated channels at membrane potentials positive to the ErevK⁺. Averaged groups (h) and their representative current responses to α-MSH (i–j)from PVN MC4R neurons voltage clamped at −55 mV in normal control (i) and in Mg²⁺ depleted (j) internal solutions. k, While zero ATP pipette solution slightly attenuated, the addition of 100 μM PI(4,5)P2 diC8 to zero ATP solution significantly increased the amplitude of the α-MSH induced depolarization recorded from PVN MC4R neurons. l, Effects of blockers of inward rectifying K⁺ channels on α-MSH (250 nM)-induced depolarization of PVN MC4R neurons compared to α-MSH alone (control) m–o, Effects of various concentrations of BaCl₂ or CsCl on the α-MSH induced depolarization measured in current clamp using similar protocol as in l. The α-MSH -induced depolarization was not blocked in higher concentrations of BaCl₂ (m–n) or CsCl (m&o), exhibiting a greater amplitude in the presence of these non-selective Kir blockers. Bars indicate mean +/−SEM, *p<0.05, ***p<0.001, unpaired t test.