Abstract
We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation (Jarvis, S. E., and Zamponi, G. W. (2001) J. Neurosci. 21, 2939-2948). Here, we have investigated the molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein beta subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to Gbeta and the N-terminal (residues 1-69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH(2)-terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both "open" and "closed" conformations during exocytosis, we produced a constitutively open form of syntaxin 1A and found that it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appeared to compete with Ggamma for the Gbeta subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein gamma subunit-like domain.
Highlights
Calcium influx through N-type calcium channels is a key step in neurotransmitter release from presynaptic nerve ter
We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation
The NH2-terminal Domains Mediate the Differential Effects of Syntaxin 1A and 1B on N-type Channel Function—We have previously reported that coexpression of syntaxin 1A, but not syntaxin 1B, with N-type (␣1B ϩ ␣2-␦1 ϩ 1b) calcium channels in tsA-201 cells results in a tonic G-protein inhibition of the channels that can be reversed following application of a strong depolarizing prepulse [26, 27], with the degree of prepulse relief being an indication of the total degree of G
Summary
All PCR reagents were purchased from Invitrogen (Rockville, MD) unless otherwise stated. The primers used were: forward, 5Ј-CCACGACCAGTGAGGAGGCCGCCGACATGCTGGAGAGTGG-3Ј; reverse, 5Ј-CCACTCTCCAGCATGTCGGCGGCCTCCTCACTGGTCGTGG-3Ј These chimeras were created in a three-step process: 1) mutagenesis to disrupt the downstream EcoRI site of the pMT2 polylinker, producing a syntaxin 1(A/B)-pMT2⌬E construct with only a single EcoRI site in the 5Ј polylinker; 2) mutagenesis to introduce a silent AgeI site at Arg108 in syntaxin 1A and Arg107 in syntaxin 1B, producing a syntaxin 1(A/ B)-pMT2⌬EϩA clone; 3) both syntaxin constructs were digested with AgeI and EcoRI and religated with the equivalent segment of the different isoform. Using XhoI and KpnI sites, the G5 insert was excised from pGEM T-Easy and subcloned in-frame into pTrcHisA This procedure was used for all His constructs in pTrcHis vectors transformed in Escherichia coli TOP10 cells (Invitrogen). The truncated band most likely arises from protease degradation in the absence of sufficient or specific protease inhibitors, it behaves the same in vitro as the full-length peptide
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