Abstract
Amphiphysin I, a member of the BAR (Bin-Amphiphysin-Rvsp) protein super family, plays a key role in clathrin-mediated endocytosis of synaptic vesicles. Amphiphysin I mediates invagination and fission of synaptic vesicles in cooperation with dynamin, which senses and facilitates membrane curvature by its BAR domain. In vitro, when present at higher concentrations, amphiphysin I can stabilize membrane curvature, generating lipid tubules, forming ring structure with dynamin, and increasing dynamin GTPase activity. In the present study, we found that amphiphysin I was cleaved to three fragments by treatment with high KCl (80 mM) in the mouse hippocampus slices. The cleavages were inhibited by pretreatment with calpain inhibitors. Calpain also cleaved amphihysin I to three fragments in vitro. We identified the three cleavage sites by mass spectrometry. Amphiphysin I was cleaved at the sites of 322, 349 and 386. Calpain-dependent cleavages of amphiphysin I can induce the liposome tubulation as the same as wild- type amphiphysin I, but it cannot form the ring structure with dynamin I under electron microscope. Moreover, transferrin uptake was inhibited by overexpression of the truncated form of amphiphysin I compared with that of wild-type amphiphysin I in COS-7 cell. These results suggest that amphiphysin I may be a substrate of calpain in presynaptic terminus and the cleavages are important for the regulation of clathrin-mediated endocytosis of synaptic vesicles. [J Physiol Sci. 2006;56 Suppl:S161]
