Constitutive activity of the dopamine (D5 ) receptor, highly expressed in CA1 hippocampal neurons, selectively reduces CaV 3.2 and CaV 3.3 currents.

BACKGROUND AND PURPOSE: CaV 3.1-3 currents differentially contribute to neuronal firing patterns. CaV 3 are regulated by G protein-coupled receptors (GPCRs) activity, but information about CaV 3 as targets of the constitutive activity of GPCRs is scarce. We investigate the impact of D5 recpetor constitutive activity, a GPCR with high levels of basal activity, on CaV 3 functionality. D5 recpetor and CaV 3 are expressed in the hippocampus and have been independently linked to pathophysiological states associated with epilepsy. EXPERIMENTAL APPROACH: Our study models were HEK293T cells heterologously expressing D1 or D5 receptor and CaV 3.1-3, and mouse brain slices containing the hippocampus. We used chlorpromazine (D1 /D5 inverse agonist) and a D5 receptor mutant lacking constitutive activity as experimental tools. We measured CaV 3 currents and excitability parameters using the patch-clamp technique. We completed our study with computational modelling and imaging technique. KEY RESULTS: We found a higher sensitivity to TTA-P2 (CaV 3 blocker) in CA1 pyramidal neurons obtained from chlorpromazine-treated animals compared with vehicle-treated animals. We found that CaV 3.2 and CaV 3.3-but not CaV 3.1-are targets of D5 receptor constitutive activity in HEK293T cells. Finally, we found an increased firing rate in CA1 pyramidal neurons from chlorpromazine-treated animals in comparison with vehicle-treated animals. Similar changes in firing rate were observed on a neuronal model with controlled CaV 3 currents levels. CONCLUSIONS AND IMPLICATIONS: Native hippocampal CaV 3 and recombinant CaV 3.2-3 are sensitive to D5 receptor constitutive activity. Manipulation of D5 receptor constitutive activity could be a valuable strategy to control neuronal excitability, especially in exacerbated conditions such as epilepsy.

LEAP2 Impairs the Capability of the Growth Hormone Secretagogue Receptor to Regulate the Dopamine 2 Receptor Signaling.

The growth hormone secretagogue receptor (GHSR) signals in response to ghrelin, but also acts via ligand-independent mechanisms that include either constitutive activation or interaction with other G protein-coupled receptors, such as the dopamine 2 receptor (D2R). A key target of GHSR in neurons is voltage-gated calcium channels type 2.2 (CaV2.2). Recently, the liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as a novel GHSR ligand, but the mechanism of action of LEAP2 on GHSR is not well understood. Here, we investigated the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on CaV2.2 function. Using a heterologous expression system and patch-clamp recordings, we found that LEAP2 impairs the reduction of CaV2.2 currents induced by ghrelin-evoked and constitutive GHSR activities, acting as a GHSR antagonist and inverse agonist, respectively. We also found that LEAP2 prevents GHSR from modulating the effects of D2R signaling on CaV2.2 currents, and that the GHSR-binding N-terminal region LEAP2 underlies these effects. Using purified labeled receptors assembled into lipid nanodiscs and Forster Resonance Energy Transfer (FRET) assessments, we found that the N-terminal region of LEAP2 stabilizes an inactive conformation of GHSR that is dissociated from Gq protein and, consequently, reverses the effect of GHSR on D2R-dependent Gi activation. Thus, our results provide critical molecular insights into the mechanism mediating LEAP2 modulation of GHSR.

Ghrelin Selectively Inhibits CaV3.3 Subtype of Low-Voltage-Gated Calcium Channels.

The mechanisms by which ghrelin controls electrical activity in the hypothalamus are not fully understood. One unexplored target of ghrelin is CaV3, responsible for transient calcium currents (T-currents) that control neuronal firing. We investigated the effect of ghrelin on CaV3 subtypes and how this modulation impacts on neuronal activity. We performed whole-cell patch-clamp recordings in primary mouse hypothalamic cultures to explore the effect of ghrelin on T-currents. We also recorded calcium currents from transiently transfected tsA201 cells to study the sensitivity of each CaV3 subtype to GHSR activation. Finally, we ran a computational model combining the well-known reduction of potassium current by ghrelin with the CaV3 biophysical parameter modifications induced by ghrelin to predict the impact on neuronal electrical behavior. We found that ghrelin inhibits native NiCl2 sensitive current currents in hypothalamic neurons. We determined that CaV3.3 is the only CaV3 subtype sensitive to ghrelin. The modulation of CaV3.3 by ghrelin comprises a reduction in maximum conductance, a shift to hyperpolarized voltages of the I-V and steady-state inactivation curves, and an acceleration of activation and inactivation kinetics. Our model-based prediction indicates that the inhibition of CaV3.3 would attenuate the stimulation of firing originating from the inhibition of potassium currents by ghrelin. In summary, we discovered a new target of ghrelin in neurons: the CaV3.3. This mechanism would imply a negative feed-forward regulation of the neuronal activation exerted by ghrelin. Our work expands the knowledge of the wide range of actions of GHSR, a receptor potentially targeted by therapeutics for several diseases.

Dopamine Receptor Type 2 and Ghrelin Receptor Coexpression Alters CaV2.2 Modulation by G Protein Signaling Cascades.

Voltage-gated calcium channels type 2.2 (CaV2.2) are activated by action potentials at presynaptic terminals, and their calcium current induces neurotransmitter release. In this context, regulating CaV2.2 is critical, and one of the most important mechanisms for doing so is through its G protein-coupled receptor (GPCR) activity. Two such GPCRs are the ghrelin (GHSR) and the dopamine type 2 (D2R) receptors. We previously demonstrated that constitutive GHSR activity reduces CaV2.2 forward trafficking and that ghrelin-induced GHSR activity inhibits CaV2.2 currents. On the other hand, dopamine-induced D2R activity also inhibits CaV2.2 currents. It has been recently shown that D2R and GHSR form heteromers in hypothalamic neurons. This interaction profoundly changes the signaling cascades activated by dopamine and is necessary for dopamine-dependent anorexia. Here we explored how D2R-GHSR coexpression in HEK293T cells modulates the effect that each GPCR has on CaV2.2. We found that D2R-GHSR coexpression reduces the inhibition of CaV2.2 currents by agonist-induced D2R activation and added a new source of basal CaV2.2 current inhibition to the one produced by GHSR solely expression. We investigated the signaling cascades implicated and found that constitutive GHSR activity, Gq protein, and Gßγ subunit play a critical role in these altered effects. Moreover, we found that the effect of D2R agonist on native calcium currents in hypothalamic neurons is reduced when both D2R and GHSR are overexpressed. In summary, our results allow us to propose a novel mechanism for controlling CaV2.2 currents involving the coexpression of two physiologically relevant GPCRs.