The G_s coupled melanocortin MC4 receptor (MC4r) leads to stimulation of cAMP formation and has been implicated in regulating energy homeostasis and penile erection. Divalent cations can modulate the function of GPCRs and recently a metal ion-biding site has been identified in the MC4r (Holst et al., 2002), highlighting the possibility its regulation by cations. Here we have characterised some functional effects of ZnCl₂ (Zn²⁺ ions) at the MC4r, in particular the regulation of Zn²⁺ effects by Ca²⁺ for both ligand-dependent and independent stimulation of cAMP formation.

All experiments were performed on HEK293 cells stably expressing the recombinant human MC4r; cAMP levels were measured directly using an ELISA (Amersham Pharmacia). Drugs were made up in HEPES buffered HBSS (with 0.5% BSA, pH 7.4) and incubated for 20mins at 37^oC.

In the presence of Ca²⁺ (1.3mM), Zn²⁺ stimulated a significant concentration-related increase in cAMP formation (4.62 ± 0.43 fold/basal at 100µM Zn²⁺, P<0.05, n=3). In the presence of the selective MC4r antagonist SHU9119 (10µM; Schioth et al., 1999), the effects of Zn²⁺ (100µM) were significantly reduced (1.48 ± 0.20 fold/basal, P<0.05, n=3), indicating that the effects were an action at the MC4r.

The effects of increasing [Ca²⁺] on the actions of Zn2+ (330µM) at the MC4r were also investigated. Ca²⁺ inhibited Zn²⁺ stimulated cAMP formation (pEC₅₀ of 3.98 ± 0.13, n=4); the inhibitory effects of Ca²⁺ were maximal at 1.3mM. There was also a component of activation, which was insensitive to the effects of Ca²⁺ at this [Zn²⁺].

Finally we suggest that Zn²⁺ is able to functionally compete with Ca²⁺ in its ability to regulate agonist (NDP-MSH and -MSH) stimulated cAMP formation (see table below). There also appears to be a further inhibitory effect of Zn²⁺ in the absence of Ca²⁺.

	1.3mM [Ca2+]e
	0µMZn2+	100µM Zn2+
	pEC50	pEC50	Emax
NDP	8.87±0.09	8.40±0.06	80.36±5.89
-MSH	8.34±0.04	6.46±0.09	81.15±6.26
	0mM [Ca2+]e
	0µMZn2+	100µM Zn2+
	pEC50	pEC50	Emax
NDP	8.35±0.18	7.64±0.23	54.38±6.82
-MSH	6.07±0.21	5.57±0.29	55.71±10.54

pEC₅₀ values and E_max values for the effects of Zn²⁺ in the presence (top) and absence (bottom) of Ca²⁺ (n=3-5).

In summary, we have shown that Ca²⁺ and Zn²⁺ ions can play a role in the regulation of cAMP formation by the MC4r in both agonist dependent and independent manners. These data may be of relevance to both the physiological role of the MC4r and to the development of assays to study its function.

Holst, B., Elling, CE., & Schwartz TW. (2002). J. Biol. Chem., 277, 47662-47770.
Schioth, HB., Muceniece, R., Mutulis, F., Bouifrouri, AA., Mutule, I., & Wikberg, JE. (1999). Neuropeptides., 33, 191-196.