Inhibition of N-Type Calcium Channels by Activation of GPR35, an Orphan Receptor, Heterologously Expressed in Rat Sympathetic Neurons

GPR35 is a G protein-coupled receptor recently "de-orphanized" using high-throughput intracellular calcium measurements in clonal cell lines expressing a chimeric G-protein α-subunit. From these screens, kynurenic acid, an endogenous metabolite of tryptophan, and zaprinast, a synthetic inhibitor of cyclic guanosine monophosphate-specific phosphodiesterase, emerged as potential agonists for GPR35. To investigate the coupling of GPR35 to natively expressed neuronal signaling pathways and effectors, we heterologously expressed GPR35 in rat sympathetic neurons and examined the modulation of N-type (Ca_V2.2) calcium channels. In neurons expressing GPR35, calcium channels were inhibited in the absence of overt agonists, indicating a tonic receptor activity. Application of kynurenic acid or zaprinast resulted in robust voltage-dependent calcium current inhibition characteristic of Gβγ-mediated modulation. Both agonist-independent and -dependent effects of GPR35 were blocked by Bordetella pertussis toxin pretreatment indicating the involvement of G_i/o proteins. In neurons expressing GPR35a, a short splice variant of GPR35, zaprinast was more potent (EC₅₀ = 1 μM) than kynurenic acid (58 μM) but had a similar efficacy (approximately 60% maximal calcium current inhibition). Expression of GPR35b, which has an additional 31 residues at the N terminus, produced similar results but with much greater variability. Both GPR35a and GPR35b appeared to have similar expression patterns when fused to fluorescent proteins. These results suggest a potential role for GPR35 in regulating neuronal excitability and synaptic release.