title: An Engineered Metal Sensor Tunes the Kinetics of Synaptic Transmission.
abstract: The Ca(2+) sensor synaptotagmin-1 (syt-1) regulates neurotransmitter release by interacting with anionic phospholipids. Here we test the idea that the intrinsic kinetics of syt-membrane interactions determine, in part, the time course of synaptic transmission. To tune the kinetics of this interaction, we grafted structural elements from the slowest isoform, syt-7, onto the fastest isoform, syt-1, resulting in a chimera with intermediate kinetic properties. Moreover, the chimera coupled a physiologically irrelevant metal, Sr(2+), to membrane fusion in vitro. When substituted for syt-1 in mouse hippocampal neurons, the chimera slowed the kinetics of synaptic transmission. Neurons expressing the chimera also evinced rapid and efficient Sr(2+) triggered release, in contrast to the weak response of neurons expressing syt-1. These findings reveal presynaptic sensor-membrane interactions as a major factor regulating the speed of the release machinery. Finally, the chimera failed to clamp the elevated spontaneous fusion rate exhibited by syt-1 KO neurons, indicating that the metal binding loops of syt-1 regulate the two modes of release by distinct mechanisms.
  
  
  CONCLUSIONS
  In calcium, synaptotagmin-1 triggers neurotransmitter release by interacting with membranes. Here, we demonstrate that intrinsic properties of this interaction control the time course of synaptic transmission. We engineered a "chimera" using synaptotagmin-1 and elements of a slower isoform, synaptotagmin-7. When expressed in neurons, the chimera slowed the rate of neurotransmitter release. Furthermore, unlike native synaptotagmin-1, the chimera was able to function robustly in the presence of strontium-a metal not present in cells. We exploited this ability to show that a key function of synaptotagmin-1 is to penetrate cell membranes. This work sheds light on fundamental mechanisms of neurotransmitter release.
authors: Department of Neuroscience, Howard Hughes Medical Institute, Molecular and Cellular Pharmacology Program, and.
authors: Chantell S
authors: http://orcid.org/0000-0001-9401-8604
authors: CS
authors: Evans
authors: Department of Neuroscience, Howard Hughes Medical Institute, Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53705-2275.
authors: David A
authors: http://orcid.org/0000-0003-3671-2336
authors: DA
authors: Ruhl
authors: Department of Neuroscience, Howard Hughes Medical Institute, Molecular and Cellular Pharmacology Program, and Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53705-2275 chapman@wisc.edu.
authors: Edwin R
authors: ER
authors: Chapman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057005:Bioengineering / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008297:Male; D008670:Metals / Q000032:analysis*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D051381:Rats; D009435:Synaptic Transmission / Q000502:physiology*
chemical_list: D008670:Metals
keywords: EPSC kinetics; exocytosis; membrane penetration; synaptic transmission; synaptotagmin