Interaction of SNARE complexes with P/Q-type calcium channels in rat cerebellar synaptosomes.
P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by omega-conotoxin MVIIC. 125I-omega-Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by omega-conotoxins GVIA or MVIIA (Ki > 1 microM), which are selective for N-type calcium channels. Solubilized 125I-omega-conotoxin MVIIC receptors were specifically recognized by antibodies directed against alpha1A calcium channel subunits, proteins known to constitute a pore with P/Q-like channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin) each immunoprecipitated a similar fraction (20-40%) of omega-conotoxin MVIIC receptors. Immunoprecipitation was not additive, suggesting that heterotrimeric (SNARE) complexes containing these three proteins interact with P/Q-type calcium channels. Immobilized monoclonal anti-syntaxin antibodies retained alpha1A calcium channel subunits of 220, 180 and 160 kDa monitored by immunoblotting with site directed antibodies. Synaptotagmin was detected in channel-associated complexes, but not synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE complexes are implicated in calcium-dependent exocytosis, a process thought to be regulated by synaptotagmin. Our results indicate that these proteins interact with P/Q-type calcium channels, which may optimize their location within domains of calcium influx.
Martin-Moutot
N
N
INSERM, Unité 374, Institut Jean Roche, Faculté de Médecine Secteur Nord, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France.
Charvin
N
N
Leveque
C
C
Sato
K
K
Nishiki
T
T
Kozaki
S
S
Takahashi
M
M
Seagar
M
M
D000818:Animals; D002121:Calcium Channel Blockers / Q000737:chemistry / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D015335:Molecular Probes; D008978:Mollusk Venoms / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D050600:SNARE Proteins; D013574:Synaptosomes / Q000378:metabolism*; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D008565:Membrane Proteins; D015335:Molecular Probes; D008978:Mollusk Venoms; D050600:SNARE Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins