Voltage-dependent G protein (Gβγ) inhibition of N-type (Ca V2.2) 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 α1B 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 Gβγ 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 α1B channels lacking these NT regions. Thus, an NT module, acting via interactions with the I-II loop, appears fundamental to such modulation.
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