Orexin-A-induced Ca Entry EVIDENCE FOR INVOLVEMENT OF TRPC CHANNELS AND PROTEIN KINASE C REGULATION*

The orexins are peptide transmitters/hormones, which exert stimulatory actions in many types of cells via the G-protein-coupled OX1 and OX2 receptors. Our previous results have suggested that low (subnanomolar) concentrations of orexin-A activate Ca entry, whereas higher concentrations activate phospholipase C, Ca release, and capacitative Ca entry. As shown here, the Ca response to subnanomolar orexin-A concentrations was blocked by activation of protein kinase C by using different approaches (12-O-tetradecanoylphorbol acetate, dioctanoylglycerol, and diacylglycerol kinase inhibition) and protein phosphatase inhibition by calyculin A. The Ca response to subnanomolar orexin-A concentrations was also blocked by Mg , dextromethorphan, and tetraethylammonium. These treatments neither affected the response to high concentrations of orexin-A nor the thapsigargin-stimulated capacitative entry. The capacitative entry was instead strongly suppressed by SKF96365. An inward membrane current activated by subnanomolar concentrations of orexin-A and the currents activated upon transient expression of trpc3 channels were also sensitive to Mg , dextromethorphan, and tetraethylammonium. Responses to subnanomolar concentrations of orexin-A (Ca elevation, inward current, and membrane depolarization) were voltage-dependent with a loss of the response around 15 mV. By using reverse transcription-PCR, mRNA for the trpc1–4 channel isoforms were detected in the CHO-hOX1-C1 cells. The expression of truncated TRPC channel isoforms, in particular trpc1 and trpc3, reduced the response to subnanomolar concentrations of orexin-A but did not affect the response to higher concentrations of orexin-A. The results suggest that activation of the OX1 receptor leads to opening of a Ca 2 permeable channel, involving trpc1 and -3, which is controlled by protein kinase C. Orexins act via two G-protein-coupled receptors called OX1R and OX2R (1, 2). They activate neurons and secretory cells by mechanisms that are not fully understood (reviewed in Refs. 3 and 4). Interaction of orexin receptors with G-proteins of the Gq/11, Gi, and Gs families has been suggested based on second messenger assays and covalent labeling of G-proteins with azido-GTP S (5). The most typical responses to orexins in neurons include increased excitability, membrane depolarization (5–10 mV), and Ca elevation (3, 4). Evidence for several different mechanisms have been proposed to explain these responses, including activation of a nonselective cation current, a decrease in K conductance, and activation of Na /Ca exchange. The orexin-stimulated Ca elevation in neurons shows an explicit dependence on extracellular Ca and is therefore likely to be due to Ca entry into cells (5–7). Activation of recombinantly expressed orexin receptors in nonexcitable cells also leads to Ca elevation (1, 8–11). High concentrations ( 10 nM) of orexins induce intracellular Ca release (9), but with lower concentrations of orexins the Ca elevations observed are dependent on extracellular Ca (8– 11) and do not appear to involve measurable discharge from stores (9). Similar results are observed upon recombinant expression of orexin receptors in neuron-like excitable cells (PC12 and Neuro2A). Activation of a novel Ca influx pathway was thus suggested. The existence of such a pathway is also indirectly suggested by the dependence of the Ca response on a negative internal membrane potential and the activation of a robust influx of Mn ions in the absence of store discharge (9). The identity of this Ca influx pathway and the mechanisms involved in its activation remain unresolved. Because the orexin-stimulated [Ca ]e-dependent Ca 2 elevation is relatively insensitive to blockers of capacitative Ca entry, such as lanthanides and 2-APB, but blocked by Ni , a different molecular entity was proposed (10). Several different pathways for receptor-activated Ca entry have also been suggested based on functional studies with other receptors. These include storeoperated Ca channels and second messenger-operated channels as well as Ca -activated Ca channels (reviewed in Ref. * This work was supported by European Union Contracts ERBBIO4CT960699 and QLG3-CT-2002-00826, the Academy of Finland, the Sigrid Jusélius Foundation, the Magnus Ehrnrooth Foundation, the Lars Hierta Foundation, the Göran Gustafsson Foundation, and the Novo Nordisk Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession number(s) AJ566614, AJ566615 and AJ566613. ¶ To whom correspondence should be addressed: A. I. Virtanen Institute for Molecular Sciences, Dept. of Neurobiology, Laboratory of Cell Biology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: karl.okerman@uku.fi. 1 The abbreviations and trivial names used are: azido-GTP S, azidoguanosine 5 -3-O-(thio)triphosphate; PKC, protein kinase C; TRPC, transient receptor potential channel; 2-APB, 2-aminoethoxydiphenyl borate; GF109203X, bisindolylmaleimide; R59022, diacylglycerol kinase inhibitor; SKF96365, 1-[ -(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride; BzATP, (2 ,3 -O-(4-benzoyl-benzoyl)ATP); DOG, dioctanoyl glycerol; TPA, 12-O-tetradecanoylphorbol-13acetate; probenecid, p-(dipropylsulfamoyl)benzoic acid; Ox-A, orexin-A; CHO-K1, Chinese hamster ovary cells; [Ca ]i, intracellular free Ca 2 concentration; ANOVA, analysis of variance; GFP, green fluorescent protein; EGFP, enhanced GFP; TEA, tetraethylammonium; Dex, dextromethorphan; RT, reverse transcription. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 3, Issue of January 21, pp. 1771–1781, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.