Depolarization-evoked secretion requires two vicinal transmembrane cysteines of syntaxin 1A.
BACKGROUND
The interactions of the voltage-gated Ca(2+) channel (VGCC) with syntaxin 1A (Sx 1A), Synaptosome-associated protein of 25 kD (SNAP-25), and synaptotagmin, couple electrical excitation to evoked secretion. Two vicinal Cys residues, Cys 271 and Cys 272 in the Sx 1A transmembrane domain, are highly conserved and participate in modulating channel kinetics. Each of the Sx1A Cys mutants, differently modify the kinetics of Cav1.2, and neuronal Cav2.2 calcium channel.


RESULTS
We examined the effects of various Sx1A Cys mutants and the syntaxin isoforms 2, 3, and 4 each of which lack vicinal Cys residues, on evoked secretion, monitoring capacitance transients in a functional release assay. Membrane capacitance in Xenopus oocytes co-expressing Cav1.2, Sx1A, SNAP-25 and synaptotagmin, which is Bot C- and Bot A-sensitive, was elicited by a double 500 ms depolarizing pulse to 0 mV. The evoked-release was obliterated when a single Cys Sx1A mutant or either one of the Sx isoforms were substituted for Sx 1A, demonstrating the essential role of vicinal Cys residues in the depolarization mediated process. Protein expression and confocal imaging established the level of the mutated proteins in the cell and their targeting to the plasma membrane.


CONCLUSIONS
We propose a model whereby the two adjacent transmembranal Cys residues of Sx 1A, lash two calcium channels. Consistent with the necessity of a minimal fusion complex termed the excitosome, each Sx1A is in a complex with SNAP-25, Syt1, and the Ca(2+) channel. A Hill coefficient >2 imply that at least three excitosome complexes are required for generating a secreting hetero-oligomer protein complex. This working model suggests that a fusion pore that opens during membrane depolarization could be lined by alternating transmembrane segments of Sx1A and VGCC. The functional coupling of distinct amino acids of Sx 1A with VGCC appears to be essential for depolarization-evoked secretion.
Cohen
Roy
R
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Marom
Merav
M
Atlas
Daphne
D
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D020746:Calcium Channels, L-Type / Q000502:physiology; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D017931:DNA Primers; D005260:Female; D007700:Kinetics; D008564:Membrane Potentials; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050827:Syntaxin 1 / Q000737:chemistry* / Q000502:physiology*; D014982:Xenopus laevis
D020746:Calcium Channels, L-Type; D017931:DNA Primers; C419385:L-type calcium channel alpha(1C); D011994:Recombinant Proteins; D050827:Syntaxin 1; D003545:Cysteine