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.
Mesh Terms:
Animals, Biotinylation, Cell Line, Cell Membrane, Chick Embryo, Down-Regulation, Guanylate Kinase, Humans, Immunoprecipitation, Large-Conductance Calcium-Activated Potassium Channel alpha Subunits, Membrane Potentials, Microscopy, Confocal, Patch-Clamp Techniques, Podocytes, Protein Binding, Protein Transport, RNA Interference, Recombinant Fusion Proteins, Transfection, Two-Hybrid System Techniques
Animals, Biotinylation, Cell Line, Cell Membrane, Chick Embryo, Down-Regulation, Guanylate Kinase, Humans, Immunoprecipitation, Large-Conductance Calcium-Activated Potassium Channel alpha Subunits, Membrane Potentials, Microscopy, Confocal, Patch-Clamp Techniques, Podocytes, Protein Binding, Protein Transport, RNA Interference, Recombinant Fusion Proteins, Transfection, Two-Hybrid System Techniques
Am. J. Physiol., Cell Physiol.
Date: Jul. 01, 2009
PubMed ID: 19403801
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