A cardiac clathrin assembly protein forms a potassium channel in planar lipid bilayers.
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 100-, 97-, and 55-kDa bands. A GTP-binding protein of approximately 25 kDa also co-purifies. The 100-kDa band was recognized by a monoclonal antibody to the gamma-adaptin of bovine brain clathrin assembly protein AP-1. A polyclonal antibody to the approximately 100-kDa doublet (alpha- and beta-adaptins) of bovine brain AP-2 did not cross-react 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 PK/PCl approximately 16 and PK/PNa approximately 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 CaCl2 (mM), inositol 1,3,4,5-tetrakisphosphate (20 microM), inositol 1,4,5-trisphosphate) (40 microM), and guanosine 5'-O-(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.
Kijima
Y
Y
Department of Molecular Biology, Vanderbilt University, Nashville Tennessee 37235.
Mayrleitner
M
M
Timerman
A P
AP
Saito
A
A
Schindler
H
H
Fleischer
S
S
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D015153:Blotting, Western; D002966:Clathrin / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D034041:Monomeric Clathrin Assembly Proteins; D009206:Myocardium / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D007295:Inositol Phosphates; D008051:Lipid Bilayers; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D015221:Potassium Channels; C439023:clathrin assembly protein AP180; D016244:Guanosine 5'-O-(3-Thiotriphosphate)