MAGI-1 interacts with Slo1 channel proteins and suppresses Slo1 expression on the cell surface.
Large conductance Ca(2+)-activated K(+) (BK(Ca)) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BK(Ca) channels using a bait construct derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues, including podocytes and the brain. The interaction between MAGI-1 and BK(Ca) channels was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays in differentiated cells of a podocyte cell line and in human embryonic kidneys (HEK)293T cells transiently coexpressing MAGI-1a and three different COOH-terminal Slo1 variants. Coexpression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knockdown of endogenous MAGI-1 expression by small interfering RNA (siRNA) in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas overexpression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BK(Ca) channels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BK(Ca) channels in the kidney and possibly in other tissues.
Ridgway
Lon D
LD
Dept. of Biology and Biochemistry, Univ. of Houston, Houston, TX 77204-5001, USA.
Kim
Eun Young
EY
Dryer
Stuart E
SE
D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002642:Chick Embryo; D015536:Down-Regulation; D051528:Guanylate Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D050199:Podocytes / Q000201:enzymology*; D011485:Protein Binding; D021381:Protein Transport; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques
D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D011993:Recombinant Fusion Proteins; D051528:Guanylate Kinases