A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release.
The function of synaptotagmin as a Ca(2+) sensor in neurotransmitter release involves Ca(2+)-dependent phospholipid binding to its two C(2) domains, but this activity alone does not explain why Ca(2+) binding to the C(2)B domain is more critical for release than Ca(2+) binding to the C(2)A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca(2+) induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca(2+) binding to the C(2)B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C(2)B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.
Dai
Han
H
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Shen
Nan
N
Araç
Demet
D
Rizo
Josep
J
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008051:Lipid Bilayers; D046210:Microfluidic Analytical Techniques; D008958:Models, Molecular; D046912:Multiprotein Complexes; D018377:Neurotransmitter Agents / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010743:Phospholipids / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050863:Synaptotagmin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D008051:Lipid Bilayers; D046912:Multiprotein Complexes; D018377:Neurotransmitter Agents; D010743:Phospholipids; D011994:Recombinant Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; D002118:Calcium