Presynaptic calcium channels regulation by ghrelin receptor (GHSR) activity.

LOPEZ SOTO JE; AGOSTI F; CABRAL A; CASTROGIOVANNI D; RODRÍGUEZ SS; MARTÍNEZ DAMONTE V; DE FRANCESCO N; PERELLO M; RAINGO J

San Diego

Congreso; Annual Meeting of Society for Neuroscience; 2013

Society for Neuroscience

Ghrelin increases appetite by acting on the central growth hormone secretagogue receptor type 1a (GHSR), the G protein coupled receptor with the highest constitutive activity known. Currently, the molecular mechanisms by which GHSR regulates neuronal functions are unclear. Within the hypothalamus, GHSR is highly enriched in synaptic terminals where presynaptic voltage gated calcium channels (CaV2.1 and CaV2.2) are also located. Thus, we tested the hypothesis that GHSR regulates presynaptic voltage gated calcium channels. In primary cultures of hypothalamic neurons transfected with GHSR, we found a 25% of inhibition of CaV native currents in response to exogenous ghrelin. Interestingly, even GHSR constitutive activity was able to inhibit native CaV currents of hypothalamic neurons. In order to study in detail if constitutive and ghrelin-evoked activity of GHSR modifies presynaptic calcium channels activity, we performed whole-cell patch-clamp recordings in HEK cells transfected with GHSR, CaV2.2 or CaV2.1 and the auxiliary subunits of the calcium channels. We found that GHSR constitutive activity inhibits CaV2.2 and CaV2.1 currents. This effect depends on the concentration of GHSR containing plasmid used and is occluded by pre-incubation with 1 μM SPA, an inverse agonist of GHSR. Moreover CaV2.2 and CaV2.1 currents are not affected by co-transfection of a mutated form of GHSR (GHSR A204E) that lacks constitutive activity. On the other hand, ghrelin inhibits CaV2.2 and CaV2.1 currents in a concentration-dependent manner (EC50=170 nM for CaV2.2 currents). We investigated the pathways involved, we found that the mechanism for basal and ghrelin-evoked inhibition are mediated by Gq proteins, but they differs in their dependency on CaVβ subunit subtype and Gβγ subunit of G protein requirement, indicating differences between the pathways. Taking together our results, we conclude that basal and evoked GHSR activity inhibits presynaptic calcium channels and this effect could contribute for the physiological effect of GHSR at presynaptic terminals in the hypothalamus. Moreover we showed for the first time the impact of GPCR constitutive activity on calcium channels activity. Supported by FONCyT (PICT2010-1589 and PICT 2011-1816 (JR) and PICT2010-1954. PICT2011-2142(MP) and by CONICET and CICPBA.