Lipid binding is required for PICK1-induced clustering of ASIC1a. A, ASIC1a and wild-type GFP-PICK1 or lipid binding-deficient mutant PICK1 2K-E were transfected into HEK293T cells. The cells were fixed and purified anti-ASIC1 antibody was used to stain ASIC1a. When transfected alone, ASIC1a, PICK1 or mutant PICK1 were all diffusely distributed throughout the cell. B, ASIC1a and GFP-PICK1 were co-transfected into HEK293T cells and stained with anti-ASIC1 antibody. Wild-type PICK1 and ASIC1a formed co-clusters in the cells. While some cells had big co-clusters in the perinuclear site of the cell (upper panel), some cells showed small co-clusters along the cellular protrusions (lower panel). C, lipid binding-deficient mutant PICK1 2K-E did not form any co-clusters with ASIC1a in cell body or cellular protrusions.