Endophilin isoforms have distinct characteristics in interactions with N-type Ca2+ channels and dynamin I

Objective

Formation of the endophilin II-Ca²⁺ channel complex is Ca²⁺-dependent in clathrin-mediated endocytosis. However, little is known about whether the other two endophilin isoforms have the same features. The present study aimed to investigate the characteristics of the interactions of all three isoforms with Ca²⁺ channels and dynamin I.

Methods

N-type Ca²⁺ channel C-terminal fragments (NCFs) synthesized with a ³H-leucine-labeled kit, were incubated with endophilin-GST fusion proteins, followed by pull-down assay. Results were counted on a scintillation counter. In addition, the different endophilin isoforms were each co-transfected with dynamin I into 293T cells, followed by flow cytometry and co-immunoprecipitation assay. Immunostaining was performed and an image analysis program was used to evaluate the overlap coefficient of cells expressing endophilin and dynamin I.

Results

All three isoforms interacted with NCF. Endophilins I and II demonstrated clear Ca²⁺-dependent interactions with NCF, whereas endophilin III did not. Co-immunoprecipitation showed that, compared to endophilin I/II, the interaction between endophilin III and dynamin I was significantly increased. Similar results were obtained from flow cytometry. Furthermore, endophilin III had a higher overlap coefficient with dynamin I in co-transfected 293T cells.

Conclusion

Endophilin isoforms have distinct characteristics in interactions with NCF and dynamin I. Endophilin III binding to NCF is Ca²⁺-independent, implying that it plays a different role in clathrin-mediated endocytosis.