Secretory carrier-associated membrane protein 5 regulates cell-surface targeting of T-type calcium channels.

Missense mutations in the human secretary carrier-associated membrane protein 5 (SCAMP5) cause a variety of neurological disorders including neurodevelopmental delay, epilepsy, and Parkinson's disease. We recently documented the importance of SCAMP2 in the regulation of T-type calcium channel expression in the plasma membrane. Here, we show that similar to SCAMP2, the co-expression of SCAMP5 in tsA-201 cells expressing recombinant Cav3.1, Cav3.2, and Cav3.3 channels nearly abolished whole-cell T-type currents. Recording of intramembrane charge movements revealed that SCAMP5-induced inhibition of T-type currents is primarily caused by the reduced expression of functional channels in the plasma membrane. Moreover, we show that SCAMP5-mediated downregulation of Cav3.2 channels is essentially preserved with disease-causing SCAMP5 R91W and G180W mutations. Hence, this study extends our previous findings with SCAMP2 and indicates that SCAMP5 also contributes to repressing the expression of T-type channels in the plasma membrane.

Electrophysiological and computational analysis of Cav3.2 channel variants associated with familial trigeminal neuralgia.

Trigeminal neuralgia (TN) is a rare form of chronic neuropathic pain characterized by spontaneous or elicited paroxysms of electric shock-like or stabbing pain in a region of the face. While most cases occur in a sporadic manner and are accompanied by intracranial vascular compression of the trigeminal nerve root, alteration of ion channels has emerged as a potential exacerbating factor. Recently, whole exome sequencing analysis of familial TN patients identified 19 rare variants in the gene CACNA1H encoding for Cav3.2T-type calcium channels. An initial analysis of 4 of these variants pointed to a pathogenic role. In this study, we assessed the electrophysiological properties of 13 additional TN-associated Cav3.2 variants expressed in tsA-201 cells. Our data indicate that 6 out of the 13 variants analyzed display alteration of their gating properties as evidenced by a hyperpolarizing shift of their voltage dependence of activation and/or inactivation resulting in an enhanced window current supported by Cav3.2 channels. An additional variant enhanced the recovery from inactivation. Simulation of neuronal electrical membrane potential using a computational model of reticular thalamic neuron suggests that TN-associated Cav3.2 variants could enhance neuronal excitability. Altogether, the present study adds to the notion that ion channel polymorphisms could contribute to the etiology of some cases of TN and further support a role for Cav3.2 channels.

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.

The ups and downs of growth hormone secretagogue receptor signaling.

The growth hormone secretagogue receptor (GHSR) has emerged as one of the most fascinating molecules from the perspective of neuroendocrine control. GHSR is mainly expressed in the pituitary and the brain, and plays key roles regulating not only growth hormone secretion but also food intake, adiposity, body weight, glucose homeostasis and other complex functions. Quite atypically, GHSR signaling displays a basal constitutive activity that can be up- or downregulated by two digestive system-derived hormones: the octanoylated-peptide ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2), which was recently recognized as an endogenous GHSR ligand. The existence of two ligands with contrary actions indicates that GHSR activity can be tightly regulated and that the receptor displays the capability to integrate such opposing inputs in order to provide a balanced intracellular signal. This article provides a summary of the current understanding of the biology of ghrelin, LEAP2 and GHSR and discusses the reconceptualization of the cellular and physiological implications of the ligand-regulated GHSR signaling, based on the latest findings.

THE INTRIGUING LIGAND-DEPENDENT AND LIGAND-INDEPENDENT ACTIONS OF THE GROWTH HORMONE SECRETAGOGUE RECEPTOR ON REWARD-RELATED BEHAVIORS.

The growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor (GPCR) highly expressed in the brain, and also in some peripheral tissues. GHSR activity is evoked by the stomach-derived peptide hormone ghrelin and abrogated by the intestine-derived liver-expressed antimicrobial peptide 2 (LEAP2). In vitro, GHSR displays ligand-independent actions, including a high constitutive activity and an allosteric modulation of other GPCRs. Beyond its neuroendocrine and metabolic effects, cumulative evidence shows that GHSR regulates the activity of the mesocorticolimbic pathway and modulates complex reward-related behaviors towards different stimuli. Here, we review current evidence indicating that ligand-dependent and ligand-independent actions of GHSR enhance reward-related behaviors towards appetitive stimuli and drugs of abuse. We discuss putative neuronal networks and molecular mechanisms that GHSR would engage to modulate such reward-related behaviors. Finally, we briefly discuss imaging studies showing that ghrelin would also regulate reward processing in humans. Overall, we conclude that GHSR is a key regulator of the mesocorticolimbic pathway that influences its activity and, consequently, modulates reward-related behaviors via ligand-dependent and ligand-independent actions.

Development of a novel fluorescent ligand of growth hormone secretagogue receptor based on the N-Terminal Leap2 region.

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently recognized as an endogenous ligand for the growth hormone secretagogue receptor (GHSR), which also is a receptor for the hormone ghrelin. LEAP2 blocks ghrelin-induced activation of GHSR and inhibits GHSR constitutive activity. Since fluorescence-based imaging and pharmacological analyses to investigate the biology of GHSR require reliable probes, we developed a novel fluorescent GHSR ligand based on the N-terminal LEAP2 sequence, hereafter named F-LEAP2. In vitro, F-LEAP2 displayed binding affinity and inverse agonism to GHSR similar to LEAP2. In a heterologous expression system, F-LEAP2 labeling was specifically observed in the surface of GHSR-expressing cells, in contrast to fluorescent ghrelin labeling that was mainly observed inside the GHSR-expressing cells. In mice, centrally-injected F-LEAP2 reduced ghrelin-induced food intake, in a similar fashion to LEAP2, and specifically labeled cells in GHSR-expressing brain areas. Thus, F-LEAP2 represents a valuable tool to study the biology of GHSR in vitro and in vivo.

Constitutive activity of the Ghrelin receptor reduces surface expression of voltage-gated Ca2+ channels in a CaVß-dependent manner.

Voltage-gated Ca2+ (CaV) channels couple membrane depolarization to Ca2+ influx, triggering a range of Ca2+-dependent cellular processes. CaV channels are, therefore, crucial in shaping neuronal activity and function, depending on their individual temporal and spatial properties. Furthermore, many neurotransmitters and drugs that act through G protein coupled receptors (GPCRs), modulate neuronal activity by altering the expression, trafficking, or function of CaV channels. GPCR-dependent mechanisms that downregulate CaV channel expression levels are observed in many neurons but are, by comparison, less studied. Here we show that the growth hormone secretagogue receptor type 1a (GHSR), a GPCR, can inhibit the forwarding trafficking of several CaV subtypes, even in the absence of agonist. This constitutive form of GPCR inhibition of CaV channels depends on the presence of a CaVß subunit. CaVß subunits displace CaVα1 subunits from the endoplasmic reticulum. The actions of GHSR on CaV channels trafficking suggest a role for this signaling pathway in brain areas that control food intake, reward, and learning and memory.