title: Origin of the voltage dependence of G-protein regulation of P/Q-type Ca2+ channels.
abstract: G-protein (Gbetagamma)-mediated voltage-dependent inhibition of N- and P/Q-type Ca(2+) channels contributes to presynaptic inhibition and short-term synaptic plasticity. The voltage dependence derives from the dissociation of Gbetagamma from the inhibited channels, but the underlying molecular and biophysical mechanisms remain largely unclear. In this study we investigated the role in this process of Ca(2+) channel beta subunit (Ca(v)beta) and a rigid alpha-helical structure between the alpha-interacting domain (AID), the primary Ca(v)beta docking site on the channel alpha(1) subunit, and the pore-lining IS6 segment. Gbetagamma inhibition of P/Q-type channels was reconstituted in giant inside-out membrane patches from Xenopus oocytes. Large populations of channels devoid of Ca(v)beta were produced by washing out a mutant Ca(v)beta with a reduced affinity for the AID. These beta-less channels were still inhibited by Gbetagamma, but without any voltage dependence, indicating that Ca(v)beta is indispensable for voltage-dependent Gbetagamma inhibition. A truncated Ca(v)beta containing only the AID-binding guanylate kinase (GK) domain could fully confer voltage dependence to Gbetagamma inhibition. Gbetagamma did not alter inactivation properties, and channels recovered from Gbetagamma inhibition exhibited the same activation property as un-inhibited channels, indicating that Gbetagamma does not dislodge Ca(v)beta from the inhibited channel. Furthermore, voltage-dependent Gbetagamma inhibition was abolished when the rigid alpha-helix between the AID and IS6 was disrupted by insertion of multiple glycines, which also eliminated Ca(v)beta regulation of channel gating, revealing a pivotal role of this rigid alpha-helix in both processes. These results suggest that depolarization-triggered movement of IS6, coupled to the subsequent conformational change of the Gbetagamma-binding pocket through a rigid alpha-helix induced partly by the Ca(v)beta GK domain, causes the dissociation of Gbetagamma and is fundamental to voltage-dependent Gbetagamma inhibition.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yu-Hang
authors.initials: YH
authors.lastname: Chen
authors.forename: Saroja D
authors.initials: SD
authors.lastname: Bangaru
authors.forename: Linling
authors.initials: L
authors.lastname: He
authors.forename: Kathryn
authors.initials: K
authors.lastname: Abele
authors.forename: Shihori
authors.initials: S
authors.lastname: Tanabe
authors.forename: Tohru
authors.initials: T
authors.lastname: Kozasa
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001703:Biophysics; D020864:Calcium Channels, N-Type / Q000502:physiology*; D002478:Cells, Cultured; D004558:Electric Stimulation / Q000379:methods; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D051528:Guanylate Kinases / Q000378:metabolism; D007313:Insecta; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors; D014982:Xenopus laevis
chemical_list: D020864:Calcium Channels, N-Type; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D021122:Protein Subunits; C496569:Grk2 protein, rat; D054769:G-Protein-Coupled Receptor Kinase 2; D051528:Guanylate Kinases; D019204:GTP-Binding Proteins