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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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Quantitation of MC4R and Kir7.1 RNA in PVN cellsSingle molecule RNA detection in sections was quantitated by counting fluorescent dots associated with individual cells (Fig. ED3). Background threshold was determined from the number of dots per cell in sections resulting from hybridization using a negative bacterial DNA control, or from hybridization of the MC4R probe to sections from the MC4R knockout mouse (a, columns 1 and 2). Threshold-subtracted dot numbers were then used to determine the % of PVN cells expressing MC4R or Kir7.1, and the % of MC4R cells expressing Kir7.1; cells were considered positive if the number of dots exceeded the mean of the negative controls by 3x std (b).
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Figure 8: Quantitation of MC4R and Kir7.1 RNA in PVN cellsSingle molecule RNA detection in sections was quantitated by counting fluorescent dots associated with individual cells (Fig. ED3). Background threshold was determined from the number of dots per cell in sections resulting from hybridization using a negative bacterial DNA control, or from hybridization of the MC4R probe to sections from the MC4R knockout mouse (a, columns 1 and 2). Threshold-subtracted dot numbers were then used to determine the % of PVN cells expressing MC4R or Kir7.1, and the % of MC4R cells expressing Kir7.1; cells were considered positive if the number of dots exceeded the mean of the negative controls by 3x std (b).

Mentions: To identify the subtype(s) of Kir channels involved in the α-MSH induced depolarization, we used a panel Kir channel blockers (Fig. 2l, and Supplementary Table 1) with previously characterized channel subtype specificity11. Additionally, we used the bee venom tertiapin Q, a blocker of Kir3.x and Kir1.1 and glibenclamide, a blocker of Kir6.x/SURx. The depolarizing effect of α-MSH was inhibited ~80% by 15 μM VU573, a selective blocker of Kir2.3, Kir3.x and Kir7.1, and blocked by the Kir7.1 blocker VU590, but not VUR5C, an inactive VU573 analogue, tertiapin Q, glibenclamide, or VU591. These data suggest that Kir7.1 generates the current underlying the α-MSH-induced depolarization in mouse PVN neurons. Kir7.1 is reported to be resistant to external BaCl2 or CsCl, at concentrations that block other Kir channels12, and the α-MSH induced depolarization persisted in the presence of BaCl2 or CsCl. The increased amplitude of this response in higher concentrations of BaCl2 (>1 mM) and CsCl (>2.5 mM), may result from non-selective blockade of other voltage sensitive outward currents (Fig. 2m–o). Additionally, Kir7.1 is more permeable to rubidium than other Kir(s)13, and when K gluconate in the patch pipette was replaced with RbCl (RbCl 130 mM and KCl 4 mM), α-MSH induced, Rb-mediated depolarization was observed and its magnitude was significantly greater than physiological K+-mediated depolarization (Extended Data Fig. 2). Using dual fluorescent in situ hybridization in sections of mouse PVN, we also determined that approximately 90% of PVN neurons expressing MC4R mRNA co-expressed Kir7.1 mRNA (Extended data Fig. 3–4).


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)

Quantitation of MC4R and Kir7.1 RNA in PVN cellsSingle molecule RNA detection in sections was quantitated by counting fluorescent dots associated with individual cells (Fig. ED3). Background threshold was determined from the number of dots per cell in sections resulting from hybridization using a negative bacterial DNA control, or from hybridization of the MC4R probe to sections from the MC4R knockout mouse (a, columns 1 and 2). Threshold-subtracted dot numbers were then used to determine the % of PVN cells expressing MC4R or Kir7.1, and the % of MC4R cells expressing Kir7.1; cells were considered positive if the number of dots exceeded the mean of the negative controls by 3x std (b).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383680&req=5

Figure 8: Quantitation of MC4R and Kir7.1 RNA in PVN cellsSingle molecule RNA detection in sections was quantitated by counting fluorescent dots associated with individual cells (Fig. ED3). Background threshold was determined from the number of dots per cell in sections resulting from hybridization using a negative bacterial DNA control, or from hybridization of the MC4R probe to sections from the MC4R knockout mouse (a, columns 1 and 2). Threshold-subtracted dot numbers were then used to determine the % of PVN cells expressing MC4R or Kir7.1, and the % of MC4R cells expressing Kir7.1; cells were considered positive if the number of dots exceeded the mean of the negative controls by 3x std (b).
Mentions: To identify the subtype(s) of Kir channels involved in the α-MSH induced depolarization, we used a panel Kir channel blockers (Fig. 2l, and Supplementary Table 1) with previously characterized channel subtype specificity11. Additionally, we used the bee venom tertiapin Q, a blocker of Kir3.x and Kir1.1 and glibenclamide, a blocker of Kir6.x/SURx. The depolarizing effect of α-MSH was inhibited ~80% by 15 μM VU573, a selective blocker of Kir2.3, Kir3.x and Kir7.1, and blocked by the Kir7.1 blocker VU590, but not VUR5C, an inactive VU573 analogue, tertiapin Q, glibenclamide, or VU591. These data suggest that Kir7.1 generates the current underlying the α-MSH-induced depolarization in mouse PVN neurons. Kir7.1 is reported to be resistant to external BaCl2 or CsCl, at concentrations that block other Kir channels12, and the α-MSH induced depolarization persisted in the presence of BaCl2 or CsCl. The increased amplitude of this response in higher concentrations of BaCl2 (>1 mM) and CsCl (>2.5 mM), may result from non-selective blockade of other voltage sensitive outward currents (Fig. 2m–o). Additionally, Kir7.1 is more permeable to rubidium than other Kir(s)13, and when K gluconate in the patch pipette was replaced with RbCl (RbCl 130 mM and KCl 4 mM), α-MSH induced, Rb-mediated depolarization was observed and its magnitude was significantly greater than physiological K+-mediated depolarization (Extended Data Fig. 2). Using dual fluorescent in situ hybridization in sections of mouse PVN, we also determined that approximately 90% of PVN neurons expressing MC4R mRNA co-expressed Kir7.1 mRNA (Extended data Fig. 3–4).

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