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A cardiac clathrin assembly protein forms a potassium channel in planar lipid bilayers.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1993 Aug 05; Vol. 268 (22), pp. 16253-8. - Publication Year :
- 1993
-
Abstract
- 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.
- Subjects :
- Adaptor Proteins, Vesicular Transport
Animals
Blotting, Western
Dogs
Electrophoresis, Polyacrylamide Gel
Guanosine 5'-O-(3-Thiotriphosphate) metabolism
In Vitro Techniques
Inositol Phosphates metabolism
Clathrin metabolism
Lipid Bilayers metabolism
Monomeric Clathrin Assembly Proteins
Myocardium metabolism
Nerve Tissue Proteins metabolism
Phosphoproteins metabolism
Potassium Channels metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 268
- Issue :
- 22
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8344911