title: G protein-gated inhibitory module of N-type (ca(v)2.2) ca2+ channels.
abstract: Voltage-dependent G protein (Gbetagamma) inhibition of N-type (CaV2.2) channels supports presynaptic inhibition and represents a central paradigm of channel modulation. Still controversial are the proposed determinants for such modulation, which reside on the principal alpha1B channel subunit. These include the interdomain I-II loop (I-II), the carboxy tail (CT), and the amino terminus (NT). Here, we probed these determinants and related mechanisms, utilizing compound-state analysis with yeast two-hybrid and mammalian cell FRET assays of binding among channel segments and G proteins. Chimeric channels confirmed the unique importance of NT. Binding assays revealed selective interaction between NT and I-II elements. Coexpressing NT peptide with Gbetagamma induced constitutive channel inhibition, suggesting that the NT domain constitutes a G protein-gated inhibitory module. Such inhibition was limited to NT regions interacting with I-II, and G-protein inhibition was abolished within alpha1B channels lacking these NT regions. Thus, an NT module, acting via interactions with the I-II loop, appears fundamental to such modulation.
authors.affiliation: Department of Biomedical Engineering and Ca2+ Signals Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Agler
authors.forename: Jenafer
authors.initials: J
authors.lastname: Evans
authors.forename: Lai Hock
authors.initials: LH
authors.lastname: Tay
authors.forename: Molly J
authors.initials: MJ
authors.lastname: Anderson
authors.forename: Henry M
authors.initials: HM
authors.lastname: Colecraft
authors.forename: David T
authors.initials: DT
authors.lastname: Yue
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000502:physiology*; D020864:Calcium Channels, N-Type; D002460:Cell Line; D004558:Electric Stimulation; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D008967:Molecular Biology / Q000379:methods; D009433:Neural Inhibition / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods; D020798:Two-Hybrid System Techniques; D015003:Yeasts
chemical_list: C500545:CACNA1B protein, human; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins