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.
Publication types
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Comparative Study
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Animals
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Calcium Channels, L-Type / chemistry
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Calcium Channels, L-Type / physiology*
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Calcium Channels, N-Type
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Cell Line
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Electric Stimulation
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GTP-Binding Protein beta Subunits / metabolism
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GTP-Binding Protein gamma Subunits / metabolism
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GTP-Binding Proteins / metabolism*
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Humans
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Ion Channel Gating / physiology*
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Membrane Potentials / physiology
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Molecular Biology / methods
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Neural Inhibition / physiology*
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Patch-Clamp Techniques / methods
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Protein Binding / physiology
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Protein Structure, Tertiary / physiology
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Protein Subunits / metabolism
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Rabbits
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Radioligand Assay / methods
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Rats
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Recombinant Fusion Proteins / metabolism
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Sequence Homology, Amino Acid
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Structure-Activity Relationship
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Transfection / methods
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Two-Hybrid System Techniques
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Yeasts
Substances
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CACNA1B protein, human
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Calcium Channels, L-Type
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Calcium Channels, N-Type
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GTP-Binding Protein beta Subunits
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GTP-Binding Protein gamma Subunits
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Protein Subunits
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Recombinant Fusion Proteins
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GTP-Binding Proteins