Characterization of two distinct modes of endophilin in clathrin-mediated endocytosis

Abstract: Endophilin, one of the main accessory proteins involved in clathrin-mediated endocytosis, interacts with other endocytic proteins, such as dynamin, by its SH3 domain. We previously reported that voltage-gated Ca2+ channels are an integral part of the synaptic vesicle (SV) endocytosis machinery through their interaction with endophilin. Formation of the endophilin–channel complex is Ca2+ dependent. A glutamate residue, E264, in endophilin is part of the primary Ca2+ sensor for Ca2+-dependent formation of the channel–endophilin complex. We proposed that endophilin exists in two distinct modes (conformations), an open mode in the absence of Ca2+, and a closed mode in the presence of Ca2+. Binding of Ca2+ switches endophilin from its open mode to the closed mode, resulting in dissociation of endophilin from other proteins. The present study is aimed at understanding the functional roles of endophilin in its two different modes, by creating two endophilin mutants, E264A and E264R, to mimic endophilin in its permanent open mode and permanent closed mode respectively. Here, we show that these two modes of endophilin have different effects on how endophilin interacts with other proteins, such as dynamin or β1-adrenergic receptors. In living cells, endophilin in its permanent closed mode does not show obvious effects on agonist-induced internalization of β1-adrenergic receptors. Endophilin, when in its permanent open mode, enhances the short-term synaptic depression in cultured hippocampal neurons, due partly to its failure to dissociate from Ca2+ channels in the presence of Ca2+. Our results show that modal switching by Ca2+ allows endophilin to regulate, more effectively, the clathrin-mediated endocytosis of SV at the nerve terminal. [Copyright &y& Elsevier]