Distinct molecular determinants govern syntaxin 1A-mediated inactivation and G-protein inhibition of N-type calcium channels

We have reported recently that syntaxin 1A mediates two effects on N-type c hannels transiently expressed in tsA-201 cells: a hyperpolarizing shift in the steady-state inactivation curve as well as a tonic inhibition of the ch annel by G-protein beta gamma subunits (Jarvis et al., 2000). Here we have examined some of the molecular determinants and factors that modulate the a ction of syntaxin 1A on N-type calcium channels. With the additional coexpr ession of SNAP25, the syntaxin 1A-induced G-protein modulation of the chann el became reduced in magnitude by similar to 50% but nonetheless remained s ignificantly higher than the low levels of background inhibition seen with N-type channels alone. In contrast, coexpression of nSec-1 did not reduce t he syntaxin 1A-mediated G-protein inhibition; however, interestingly, nSec- 1 was able to induce tonic G-protein inhibition even in the absence of synt axin 1A. Both SNAP25 and nSec-1 blocked the negative shift in half-inactiva tion potential that was induced by syntaxin 1A. Activation of protein kinas e C via phorbol esters or site-directed mutagenesis of three putative PKC c onsensus 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. Ho wever, in the S896E and S898E mutants, or after PKC-dependent phosphorylati on 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 ga ting and its modulation by G-proteins.