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The Journal of Neuroscience, November 15, 1998, 18(22):9163-9170

G-Protein -Subunit Specificity in the Fast Membrane-Delimited Inhibition of Ca²⁺ Channels

David E. García^{1, 5}, Bin Li³, Rafael E. García-Ferreiro⁵, Erick O. Hernández-Ochoa⁵, Kang Yan⁴, Narasimhan Gautam⁴, William A. Catterall³, Ken Mackie^{1, 2}, and Bertil Hille¹

Departments of ¹ Physiology and Biophysics, ² Anesthesiology, and ³ Pharmacology, University of Washington, Seattle, Washington 98195, ⁴  Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, and ⁵ Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Apartado Postal 70250, C.P. 04510, México, Distrito Federal México

We investigated which subtypes of G-protein  subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein  subunits. G₁ and G₂ strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The G₅ subunit produced much weaker effects than G₁ and G₂, whereas G₃ and G₄ were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, G₁ or G₂ coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a ₁ subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the G₅ subunit interacted weakly, and G₃ and G₄ failed to interact. Together, these results suggest that G₁ and/or G₂ subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel ₁ subunit is a principal receptor for this inhibition.

Key words: G-proteins; calcium channel; norepinephrine; yeast 2-hybrid; sympathetic neurons; ion channel modulation

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Copyright © 1998 Society for Neuroscience  0270-6474/98/18229163-08$05.00/0

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