Structural/functional analysis of synaptotagmin 1 in synaptic transmission using hippocampal autapses

Abstract

Synaptotagmin 1 is a presynaptic vesicular protein, which has been implicated as a putative Ca2+ sensor for fast neurotransmitter release in central nervous system. Structurally, it possesses two Ca2+ binding C2 domains in its cytosolic C-terminal. The biochemical interaction of synaptotagmin 1 with other molecules has been extensively addressed in recent studies, while the exact physiological roles of the two C2 domains and the mechanism of action of synaptotagmin 1 in exocytosis are still poorly understood.To answer these questions better, a systematic study combining electrophysiological and molecular biological approaches was performed. The synaptic transmission of the cultured synaptotagmin 1 null murine hippocampal neurons transfected with the mutated synaptotagmin 1 was evaluated with standard whole-cell patch clamp electrophysiology. To take a closer look at how the structure of the C2 domains is important for the function of synaptotagmin 1, we made a series of the C2 domain mutations. Truncation of either C2 domain, neutralization of the Ca2+ binding aspartate sites (namely, D172, 230, 232A; D303, 363, 365A) in either C2 domain and replacement of either C2 domains with the C2 domain from other synaptotagmin isoform (i.e. synaptotagmin 7) all suggested that the two C2 domains are non redundant in synaptic transmission. Specifically, the C2A domain is related to readily releasable pool control and the regulation of fast release, whereas the C2B domain is essential for fast release.Synaptotagmin 1 has a set of hydrophobic residues (M173, F231, 234; V304, Y364, I367) in the C2 domains, which have been biochemically shown to interact with the phospholipid plasma membrane. We replaced these residues with Tryptophans with the idea of introducing greater hydrophobicity into the domains so as to correlate its physiological consequences on its phospholipid binding. These mutations result in enhancement in the release probability and apparent Ca2+ sensitivity. Furthermore, the polybasic residues in the C2B domain of synaptotagmin 1 have been shown to have multiple interactions (i. e. with another copy of synaptotagmin, PIP2 in plasma membrane, AP-2 and Ca2+ channels, etc). These interactions can be abolished by neutralizing these residues (K326, 327A). The physiological analysis of the phenotype of synaptotagmin 1 with K326, 327A mutation shows that the neurotransmitter release does not get abolished, rather it is reduced, which correlates with a corresponding decrease in in vitro Ca2+ dependent synaptotagmin 1-phospholipid binding. Interestingly, this K326, 327A phenotype bears striking similarity to the previously described synaptotagmin 1 C2A domain R233Q mutation. To further investigate this phenomenon, the corresponding site of the R233Q mutation in the C2B domain was mutated (K366Q), however, the latter one showed a wild type like behavior both in vitro and in vivo. Taken together, the asymmetrical distribution of these basic residues for vesicle release control in the two C2 domains indicates that the two C2 domains interact with plasma membrane upon coming Ca2+ in different orientation.Overall, these C2 domain mutation studies suggest that the Ca2+ dependent synaptotagmin 1-phospholipid interaction is critical for the efficiency of synaptic transmission, which ultimately supports the general notion that synaptotagmin 1 must essentially interact with the plasma membrane so as to enable vesicle fusion.