Synaptotagmins are membrane trafficking proteins that are characterized by two C-terminal C2 domains (C2A and C2B). Eight of the seventeen mammalian isoforms serve as calcium sensors that trigger exocytosis; Syt1 is predominantly involved in rapid neurotransmitter release while Syt7 triggers slower secretion events such as insulin exocytosis. Our previous work has shown that the C2A domain of human Syt7 has association and dissociation rates that are ~2- and ~60-fold slower than the corresponding domain of Syt1 (Brandt DS, Biochemistry; 51(39):7654-7664.). In the present study, structural origins of these kinetic differences were investigated using site-directed mutagenesis and stopped-flow fluorescence spectroscopy. Rates of protein-lipid association and dissociation were measured for liposomes either approximating the inner leaflet composition of a biological plasma membrane (PM) or simplified to a 3:1 ratio of phosphatidylcholine and phosphatidylserine (PC/PS). We find that phenylalanine-167 of Syt7 is responsible to a significant extent for this domain's stronger lipid affinity, as mutation of this residue to methionine (which is found at the corresponding position in Syt1) produced dissociation rates between 2- to 10-fold faster than wild-type Syt7 C2A without affecting association kinetics. Intriguingly, the dissociation rate of wild-type Syt7 C2A, but not F167M, was found to be dependent on the presence of additional background lipids such as cholesterol and sphingomyelin. These preliminary results suggest that F167 may serve as a secondary lipid anchor for Syt7 providing substantial favorable membrane interactions, and possibly as a sensor for membrane cholesterol content.

Investigating the differences in lipid-binding affinities and kinetics between C2A domains of synaptotagmins 1 and 7

248th National Meeting (2014)

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