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G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons.

Ghamari-Langroudi M, Digby GJ, Sebag JA, Millhauser GL, Palomino R, Matthews R, Gillyard T, Panaro BL, Tough IR, Cox HM, Denton JS, Cone RD - Nature (2015)

Bottom Line: Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding.Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN.Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology &Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

ABSTRACT
The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

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α-MSH depolarizes and AgRP hyperpolarizes hypothalamic PVN MC4R neurons by regulating Kir7.1a–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.
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Figure 2: α-MSH depolarizes and AgRP hyperpolarizes hypothalamic PVN MC4R neurons by regulating Kir7.1a–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.

Mentions: The currents underlying MC4R mediated depolarization of PVN neurons were then characterized with current–voltage (I–V) analysis in external 20 mM K+. Using whole cell recording from voltage clamped neurons pre-treated with 0.5 μM tetrodotoxin (TTX), 200 μM picrotoxin (PTX) and 1 mM kynurenic acid (KYN), current responses of PVN neurons to voltage ramps (−120 to −20 mV for 2 s) were used to generate the I–V relationships of the α-MSH induced response (Fig. 2a–c). α-MSH significantly (33%) decreased cell membrane conductance from 5.7 +/−1.0 nS in control to 3.8 +/− 0.8 nS (Fig. 2g), with current generated by α-MSH being linear from −120 to −60 mV that rectified inwardly at membrane potentials negative to its reversal of polarity. Its reversal potential was near −48.1 +/− 3.4 mV in the presence of 20 mM external K+, with an estimated reversal potential of K+ near −53.5 mV (Nernst equation). These results, in addition to those performed in 3.1 mM external [K+] (estimated ErevK+= ~−98 mV, not shown), suggest that α-MSH generates an inward current by closure of a steady state K+ mediated inward rectifier current, as in ARC neurons8.


G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons.

Ghamari-Langroudi M, Digby GJ, Sebag JA, Millhauser GL, Palomino R, Matthews R, Gillyard T, Panaro BL, Tough IR, Cox HM, Denton JS, Cone RD - Nature (2015)

α-MSH depolarizes and AgRP hyperpolarizes hypothalamic PVN MC4R neurons by regulating Kir7.1a–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.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: α-MSH depolarizes and AgRP hyperpolarizes hypothalamic PVN MC4R neurons by regulating Kir7.1a–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.
Mentions: The currents underlying MC4R mediated depolarization of PVN neurons were then characterized with current–voltage (I–V) analysis in external 20 mM K+. Using whole cell recording from voltage clamped neurons pre-treated with 0.5 μM tetrodotoxin (TTX), 200 μM picrotoxin (PTX) and 1 mM kynurenic acid (KYN), current responses of PVN neurons to voltage ramps (−120 to −20 mV for 2 s) were used to generate the I–V relationships of the α-MSH induced response (Fig. 2a–c). α-MSH significantly (33%) decreased cell membrane conductance from 5.7 +/−1.0 nS in control to 3.8 +/− 0.8 nS (Fig. 2g), with current generated by α-MSH being linear from −120 to −60 mV that rectified inwardly at membrane potentials negative to its reversal of polarity. Its reversal potential was near −48.1 +/− 3.4 mV in the presence of 20 mM external K+, with an estimated reversal potential of K+ near −53.5 mV (Nernst equation). These results, in addition to those performed in 3.1 mM external [K+] (estimated ErevK+= ~−98 mV, not shown), suggest that α-MSH generates an inward current by closure of a steady state K+ mediated inward rectifier current, as in ARC neurons8.

Bottom Line: Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding.Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN.Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology &Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

ABSTRACT
The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

Show MeSH
Related in: MedlinePlus