PT  - JOURNAL ARTICLE
AU  - Scott E. Jarvis
AU  - Gerald W. Zamponi
TI  - Distinct Molecular Determinants Govern Syntaxin 1A-Mediated Inactivation and G-Protein Inhibition of N-Type Calcium Channels
AID  - 10.1523/JNEUROSCI.21-09-02939.2001
DP  - 2001 May 01
TA  - The Journal of Neuroscience
PG  - 2939--2948
VI  - 21
IP  - 9
4099  - http://www.jneurosci.org/content/21/9/2939.short
4100  - http://www.jneurosci.org/content/21/9/2939.full
SO  - J. Neurosci.2001 May 01; 21
AB  - 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 βγ 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 ∼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.