We have reported recently that syntaxin 1A mediates two effects on N-type channels transiently expressed in tsA-201 cells: a hyperpolarizing shift in the steady-state inactivation curve as well as a tonic inhibition of the channel by G-protein betagamma subunits (Jarvis et al., 2000). Here we have examined some of the molecular determinants and factors that modulate the action of syntaxin 1A on N-type calcium channels. With the additional coexpression of SNAP25, the syntaxin 1A-induced G-protein modulation of the channel became reduced in magnitude by approximately 50% but nonetheless remained significantly higher than the low levels of background inhibition seen with N-type channels alone. In contrast, coexpression of nSec-1 did not reduce the syntaxin 1A-mediated G-protein inhibition; however, interestingly, nSec-1 was able to induce tonic G-protein inhibition even in the absence of syntaxin 1A. Both SNAP25 and nSec-1 blocked the negative shift in half-inactivation potential that was induced by syntaxin 1A. Activation of protein kinase C via phorbol esters or site-directed mutagenesis of three putative PKC consensus sites in the syntaxin 1A binding region of the channel (S802, S896, S898) to glutamic acid (to mimic a permanently phosphorylated state) did not affect the syntaxin 1A-mediated G-protein modulation of the channel. However, in the S896E and S898E mutants, or after PKC-dependent phosphorylation of the wild-type channels, the susceptibility of the channel to undergo shifts in half-inactivation potential was removed. Thus, separate molecular determinants govern the ability of syntaxin 1A to affect N-type channel gating and its modulation by G-proteins.