Determination of phosphoinositide binding to K(+) channel subunits using a protein-lipid overlay assay.
Phosphoinositides are an important component of the cell as they have a variety of roles that include cytoskeleton regulation, generation of second messengers, endosome trafficking, membrane transport and regulation of ion channels. The direct interaction between phosphatidylinositol-4,5-bisphosphate (PIP(2)) and various inwardly rectifying potassium channels has been shown in recent years. Most of these studies have used existing electrophysiological methods. In this review, we describe a rapid and convenient biochemical assay that can be used to show direct binding of potassium channel subunits to anionic phospholipids. This method has been used to demonstrate the differences in affinity between members of the Kir3.0 family, where only the cytoplasmic C-terminal Kir3.1 domain and the N- and C-terminal domains of Kir3.4 have the ability to bind to anionic phospholipids.
Thomas
Alison M
AM
Department of Medicine, University College London, UK.
Tinker
Andrew
A
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000502:physiology; D029968:Escherichia coli Proteins / Q000378:metabolism; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D054340:Receptors, KIR / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D029968:Escherichia coli Proteins; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D015221:Potassium Channels; D021122:Protein Subunits; D054340:Receptors, KIR; D011994:Recombinant Proteins