Ghrelin receptor (GHSR) and dopamine receptor type 2 (D2R) co-expression modifies each receptor's effects on voltage gated calcium channel CaV2.2

CORDISCO GONZALEZ, SANTIAGO; RODRÍGUEZ, SILVIA SUSANA; MUSTAFÁ, EMILIO ROMÁN; RAINGO, JESICA

Chicago

Congreso; 2019 Neuroscience Meeting Planner, Society for Neuroscience; 2019

Society for Neuroscience

Presynaptic CaV2.2 are activated by action potentials, and their calcium current induces neurotransmitter release. In this context, the regulating of CaV2.2 activity is critical, and one of the most important mechanisms for doing so is through G-protein coupled receptor (GPCR) activity. Two members of GPCRs able to modulate Cav activity are the ghrelin receptor (GHSR) and the dopamine receptor type 2 (D2R). We have previously demonstrated that GHSR constitutive activity reduces CaV2.2 trafficking to the plasma membrane and that ghrelin-induced GHSR activity inhibits CaV2.2 current. On the other hand, dopamine-induced D2R activity also inhibits CaV2.2 current. It has been recently shown that D2R and GHSR are able to hetero-dimerize in hypothalamic neurons. Here we explore how co-expression of GHSR and D2R modulates the effect that each GPCR has individually on CaV2.2. We found that GHSR-D2R co-expression increases the basal inhibition of CaV2.2 by GHSR constitutive activity, since less GHSR is needed to reduce CaV2.2 current when D2R is co-transfected. We tested if this effect is trough GHSR constitutive activity using as a tools an inverse agonist SPA (Substance-P analog) and a mutated version of GHSR (A204E) lacking of constitutive activity, and we were able to occlude the inhibitory effect. Next, we explore the signaling cascade implied in this effect on CaV2.2 and we found that co-expression of a Gq dominant negative mutant or co-expression of t Gβγ buffer peptide (Mas-GRK2-ct) completely block the reduction in the amplitude of CaV2.2 currents. By contrast, the acute inhibitory effect of ghrelin on CaV2.2 current is unaffected by GHSR-D2R co-expression. Meanwhile, GHSR-D2R co-expression decreases inhibition of CaV2.2 by dopamine-evoked D2R activity (increase in EC50), since a higher dopamine concentration is needed to inhibit CaV2.2 current when GHSR is co-transfected. This last effect depends on GHSR constitutive activity, since it is occluded by pre-incubation with SPA, and is coupled to Gq protein. Currently, we are exploring if this novel effect of GHSR-D2R hetero-dimerization has an impact on native calcium currents on hypothalamic primary cultures.