MAGI-1 interacts with Slo1 channel proteins and suppresses Slo1 expression on the cell surface
| Autor: | Eun Young Kim, Lon D. Ridgway, Stuart E. Dryer |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
BK channel
Patch-Clamp Techniques Physiology Renal glomerulus Recombinant Fusion Proteins Down-Regulation Chick Embryo Plasma protein binding Membrane-associated guanylate kinase Transfection Cell Line Membrane Potentials Cell membrane Two-Hybrid System Techniques medicine Animals Humans Immunoprecipitation Biotinylation Large-Conductance Calcium-Activated Potassium Channel alpha Subunits Membrane potential Microscopy Confocal biology Podocytes Cell Membrane Cell Biology Cell biology Transport protein Protein Transport medicine.anatomical_structure biology.protein RNA Interference Membrane Transporters Ion Channels and Pumps Guanylate Kinases Protein Binding |
| Zdroj: | American Journal of Physiology-Cell Physiology. 297:C55-C65 |
| ISSN: | 1522-1563 0363-6143 |
| DOI: | 10.1152/ajpcell.00073.2009 |
| Popis: | Large conductance Ca2+-activated K+(BKCa) 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 BKCachannels 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 BKCachannels 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 BKCachannels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BKCachannels in the kidney and possibly in other tissues. |
| Databáze: | OpenAIRE |
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