Several SNARE proteins specifically interact with CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels by binding to a synaptic protein interaction site (synprint) located within the intracellular loop connecting domains II and III (LII-III) of the channels. This interaction not only allows secretory vesicles docking to the plasma membrane near the pathway for Ca2+ entry, the signal that triggers exocytotic neurotransmitter release, but also, under certain conditions, shifts the voltage dependence of steady-state inactivation towards more negative membrane potentials. Besides the important anchoring function of the synprint site in the functional interaction SNARE proteins-CaV2.1 channels, the involvement of other molecular domains has been proposed including the alpha1A first intracellular loop (LI-II). The molecular mechanism by which LI-II influences CaV2.1-SNAREs functional interaction is unknown. It has been suggested that conformational changes at LI-II produced by different regulatory beta subunits not only determine the degree of voltage-dependent inactivation but also the extent of a Ca2+-dependent inactivation component (mediated by the binding of Ca2+-calmodulin to a IQ-like motif in alpha1A C-terminal domain). We have now analyzed CaV2.1 channel modulation by syntaxin-1A in the presence of functionally different ancillary beta subunits (beta2a or beta3) and using different human alpha1A constructs containing either a LI-II partial deletion or mutations in the IQ-like region. Our results reveal that regulation by syntaxin-1A of CaV2.1 channel activity requires both the integrity of alpha1A LI-II and the lack of a Ca2+-dependent component in the channel steady-state inactivation. Supported by Spanish Ministry of Economy and Competitiveness (Grant SAF2012-31089, FEDER Funds, Fondo de Investigación Sanitaria, Instituto Carlos III, Spain (RIC RD12/0042/0014, Red HERACLES).