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The Journal of Neuroscience, October 18, 2006, 26(42):10658-10666; doi:10.1523/JNEUROSCI.2609-06.2006

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Cellular/Molecular
The Orexin OX₁ Receptor Regulates Ca²⁺ Entry via Diacylglycerol-Activated Channels in Differentiated Neuroblastoma Cells

Johnny Näsman,¹ Genevieve Bart,¹ Kim Larsson,¹ Lauri Louhivuori,¹ Hanna Peltonen,¹ and Karl E. O. Åkerman^1,2

¹A. I. Virtanen Institute for Molecular Sciences, Department of Neurobiology, University of Kuopio, FIN-70211 Kuopio, Finland, and ²Department of Neuroscience, Uppsala University, Biomedical Center, S-75123 Uppsala, Sweden

Correspondence should be addressed Johnny Näsman, A. I. Virtanen Institute for Molecular Sciences, P.O. Box 1627, FIN-70211 Kuopio, Finland. Email: johnny.nasman{at}uku.fi

We studied the cellular response to orexin type 1 receptor (OX₁R) stimulation in differentiated IMR-32 neuroblastoma cells. In vitro differentiation of IMR-32 cells with 5-bromo-2'-deoxyuridine leads to a neuronal phenotype with long neurite extensions and an upregulation of mainly N-type voltage-gated calcium channels. Transduction of differentiated IMR-32 cells with baculovirus harboring an OX₁R–green fluorescent protein cDNA fusion construct resulted in appearance of fluorescence that was confined mainly to the plasma membrane in the cell body and to neurites. Application of orexin-A to fluorescent cells led to an increase in intracellular free Ca²⁺ concentration, [Ca²⁺]_i. At low nanomolar concentrations of orexin-A, the response was reversibly attenuated by removal of extracellular Ca²⁺, by application of a high concentration (10 mM) of Mg²⁺, and by the pharmacological channel blocker dextromethorphan. A diacylglycerol, dioctanoylglycerol, but not thapsigargin or depolarization with potassium, mimicked the OX₁R response with regard to Mg²⁺ sensitivity. A reverse transcription-PCR screening identified mRNAs for all transient receptor potential canonical (TRPC) channels, including TRPC3, TRPC6, and TRPC7, which are known to be activated by diacylglycerol. Expression of a dominant-negative TRPC6 channel subunit blunted the responses to both dioctanoylglycerol and OX₁R stimulation. The results suggest that the OX₁R activates a Ca²⁺ entry pathway that involves diacylglycerol-activated TRPC channels in neuronal cells.

Key words: baculovirus; calcium; differentiation; neuroblastoma; orexin; TRP channel

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Received Oct. 19, 2005; revised Aug. 16, 2006; accepted Aug. 16, 2006.

Correspondence should be addressed Johnny Näsman, A. I. Virtanen Institute for Molecular Sciences, P.O. Box 1627, FIN-70211 Kuopio, Finland. Email: johnny.nasman{at}uku.fi

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