A Cardiac Clathrin Assembly Protein Forms a Potassium Channel in

A novel clathrin assembly protein (designated cardiac AP-3) has been isolated from dog heart which forms a K+ channel in planar lipid bilayers. AP-3 facilitated the in vitro formation of clathrin cages, which is diagnostic for clathrin assembly proteins. AP-3 consists mainly of loo-, 97-, and 55-kDa bands. A GTPbinding protein of -25 kDa also co-purifies. The 100kDa band was recognized by a monoclonal antibody to the y-adaptin of bovine brain clathrin assembly protein AP-1. A polyclonal antibody to the -100-kDa doublet (a- and p-adaptins) of bovine brain AP-2 did not crossreact with the purified protein. Western blot analysis of cardiac subcellular fractions showed that anti-AP-1 immunoreactivity was strongest in a sarcolemma-enriched fraction. Little immunoreactivity was detected in other cardiac subfractions, including sarcoplasmic reticulum, intercalated discs, and mitochondria. When reconstituted into planar lipid bilayers, AP-3 displays ion channel activity. Permeability ratios were PKjPcl -16 and P~PN. -3, indicating a cation-selective channel somewhat selective for K+ versus Na+. The K+ channel displays several subconductance states (9 and 12 picosiemens in the main) and was blocked by CaClz (mM), inositol 1,3,4,5-tetrakisphosphate (20 p~), inositol 1,4,5-trisphosphate (40 MM), and guanosine 5‘-0(3-thiotrisphosphate) (mM). Thus, the cardiac AP-3 appears to act as a K+ channel modulated by inositol polyphosphates and a small GTP-binding protein.