Distinct Ca2+ and Sr2+ binding properties of synaptotagmins. Definition of candidate Ca2+ sensors for the fast and slow components of neurotransmitter release.
Ca(2+)-dependent neurotransmitter release consists of at least two components: a major fast component that is insensitive to Sr2+ and a minor slow component that is potentiated by Sr2+ (Goda, Y., and Stevens, C. F. (1994) Proc. Natl. Acad. U. S. A. 91, 12942-12946). These results suggest that at least two Ca2+ sensors act in synaptic vesicle fusion with distinct Ca2+ and Sr2+ binding properties. We have now investigated the relative Ca2+ and Sr2+ binding activities of synaptotagmins to evaluate their potential roles as Ca2+ sensors for the fast and slow components. Our results demonstrate that the first C2 domains of synaptotagmins I, II, III, V, and VII have very similar Ca2+ requirements for phospholipid binding (range of EC50 = 2.6 microM to 5.0 microM), but distinct Sr2+ requirements (EC50 range = 23 microM to 133 microM); synaptotagmins I and II had the lowest Sr2+ affinity, and synaptotagmin III the highest Sr2+ affinity. Purified synaptotagmin I from bovine brain exhibited similar properties as its recombinant first C2 domain, suggesting that the first C2 domain fully accounts for its Ca(2+)-dependent phospholipid binding properties. Sr2+ was unable to trigger syntaxin binding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C2 domains have distinct Sr2+ binding properties. They support the hypothesis that synaptotagmins localized on the same vesicle perform distinct functions, with synaptotagmins I and II serving as candidate Ca2+ sensors for the fast component in release and synaptotagmin III for the slow component.
Li
C
C
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Davletov
B A
BA
Südhof
T C
TC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002417:Cattle; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D050765:Qa-SNARE Proteins; D013324:Strontium / Q000378:metabolism*; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D010743:Phospholipids; D050765:Qa-SNARE Proteins; D050863:Synaptotagmin I; D050857:Synaptotagmins; D002118:Calcium; D013324:Strontium