Synaptotagmin's Role as the Ca2+ Sensor in Regulated Exocytosis

Abstract

Exocytosis of neurotransmitters is triggered by the initial influx of Ca2+. Synaptotagmin I is known to bind Ca2+ and the phospholipid membrane to modulate this process. The mechanism, however, for this information transduction is not well known. We seek to understand how this information is conveyed through the protein. A single point mutation, Y180F, has been made to correlate with a mutant that has been seen in vivo to display diminished physiological function. This mutant will be utilized as a probe as we seek to ascertain the mechanism of the signal transduction. The Y180F mutation is located in the binding pocket of the first C2 domain of synaptotagmin. It was hypothesized that this mutation abolishes the hydrogen bonding potential between this position and His237 and that the lack of hydrogen bonding will lead to a drastic decrease in stability in the binding pocket. This mutation is predicted to manifest itself as a reduction in the domain's calcium ion affinity. To test these predictions, Ca2+ and phospholipid binding assays for wild type C2A and Y180F will be carried out and monitored via steady state fluorescence as well as protein denaturation assays. Partition functions will be derived to quantify the results so a thermodynamic comparison can be carried out.