Inositol polyphosphate receptor and clathrin assembly protein AP-2 are related proteins that form potassium-selective ion channels in planar lipid bilayers.

Abstract

Inositol polyphosphate receptor and clathrin assembly protein AP-2 are related proteins that form potassium-selective ion channels in planar lipid bilayers (receptor-mediated Clathrin-coated vesicles similar to fraction D described by Keen et al. (11) were prepared from three or four whole bovine brains.Clathrin and AP-2 were partially purified from the coated vesicles essentially as described by Keen et al. (11). Briefly, coated vesicles (1.5 g of protein) were extracted with 0.5 M Tris Cl (200 ml) in the absence of detergents. The extract was precipitated in 50% ammonium sulfate, resuspended in 15-20 ml of buffer B (0.5 M Tris Cl, pH 7.0/50 mM NaMes/0.5 mM EGTA/0.25 mM MgCl2/10% glycerol), applied to an HPLC gel exclusion column (2.1 x 60 cm TSK G-4000SW or TSK G-3000 SW column; TosoHaas, Philadelphia) equilibrated in buffer B, and eluted at 2.5 ml/min, collecting 3-ml fractions. Fractions from the G-4000 column enriched in clathrin (fractions 33-40) were pooled, dialyzed overnight against buffer C (50 mM Tris Cl, pH 8.3/1 MM EDTA/1 mM dithiothreitol/ 10% glycerol), concentrated to about 1 ml by ultrafiltration (Centriprep 30; Amicon), frozen in liquid nitrogen, and stored at -80TC. Fractions from the G-3000 or G4000 column enriched in AP-2 (typically fractions 42-49 of the G-4000 column) were pooled and dialyzed overnight against 2-4 liters of buffer C. In some preparations, the dialysate was concentrated to about 1 ml by ultrafiltration; we refer to this 8976The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Characterization of a high‐affinity Ins‐P4 (inositol 1,3,4,5‐tetrakisphosphate) receptor from brain by an anti‐peptide antiserum

“…Previously Theibert et al [12] have described protein complexes occurring in brain tissue which bind Ins-P4 and with similar affinity Ins-Ps. One of these proteins displays K + channel activity [13]. The recently reported 104 kDa protein with Ins-P4 binding activity found in pig platelets [14] suggests the possibility that in non-brain, peripheral tissue Ins-P4 acts via some other Ins-P4 receptor proteins.…”

Biochemical and Functional Characterization of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinases

“…The role of amphiphysin-1 in clathrin-mediated endocytosis during synaptic vesicle reformation involves interactions with synaptojanin-1 (an inositol-polyphosphate 5-phosphatase), clathrin, and AP-2 (28,(41)(42)(43)(44)(45)(46). Interestingly, AP-2 has been characterized in vitro as an IP 6 -binding protein (47)(48)(49), and IP 6 alters the in vitro binding of AP-2 to synaptotagmin (50). Based on these reports, it was possible that scRvs167 and scRvs161 might have some function in the IP pathway.…”

Myelin Proteolipid Protein (PLP), but Not DM-20, Is an Inositol Hexakisphosphate-binding Protein

“…Partial amino acid sequencing of one protein has revealed that it is clathrin assembly protein 2 (AP-2), which is possibly an essential protein in the endocytotic or recycling pathway of all cells (17)(18)(19)(20) (18) is 120 nM. Dependence of the InsP X binding on the salt concentration in the assay system seems to account for this difference.…”

“…Several proteins involved in intracellular vesicular transport have been identified as InsP 6 -binding proteins. A clathrin assembly protein, AP-2 (17)(18)(19)(20), may be an essential protein in the endocytotic recycling pathway of all cells (21). Binding of InsP 6 inhibits the clathrin assembly me-diated by AP-2 (22) and AP-3, a synapse-specific clathrin assembly protein (23,24).…”