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 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.
Jarvis
S E
SE
Department of Physiology and Biophysics, Neuroscience and Smooth Muscle Research Groups, University of Calgary, Calgary, T2N 4N1, Canada.
Zamponi
G W
GW
D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics; D000954:Antigens, Surface / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D020864:Calcium Channels, N-Type / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000096:biosynthesis / Q000134:cerebrospinal fluid* / Q000378:metabolism* / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism; D013329:Structure-Activity Relationship; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D014162:Transfection; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D020864:Calcium Channels, N-Type; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; C494208:SNAP25 protein, human; C494212:STX1A protein, human; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D011493:Protein Kinase C; D019204:GTP-Binding Proteins