Crystallization Technique for Localizing Ion Channels in Living Cells
Part of the
Methods in Pharmacology and Toxicology
book series (MIPT)
A detailed mechanistic understanding of ion-channel function has been achieved over the last 40 years, propelled in the last 15 years by advances in patch-clamp technology and most recently by determination of channel structures and their manipulation. From the pharmacological perspective, perhaps as important as knowing how channels work, is knowing where they are—both where in tissues, and ultimately, where in individual cells. Because ion channels are integral membrane proteins, their localization can be studied using established general approaches. Immunofluorescent labeling has been widely used to map cloned Ca2+ (1), Na+(2), and K+ (3) channels, for which antibodies have been raised against the channel protein. Different variations of channel immunolabeling, including immunogold (4) (Na+ channels) or radiolabel staining are available in this arsenal. A less general but specific technique commonly used with ion channels is to use high-affinity toxins, either fluorescently or radioactively labeled, that specifically bind to the channel pore or regulatory subunits. Among these are scorpion toxins to map Na+ channels (5), Σ-conotoxin to map Ca2+ channels (6), α-dendrotoxin to map voltage-gated channels (7) and α-bungarotoxin (8) to map nicotinic ACh receptors. Such techniques, in addition to being expensive and time consuming, generally require tissue fixation, and are not amenable to living cells. Green fluorescent protein (GFP) (9) based technology is now revolutionizing the localization of proteins, including ion channels (see Part 2 of this book). Attaching GFP to N or C termini of the protein of interest permits visualization in living or fixed preparations, generally without significantly affecting channel function.
KeywordsXenopus Oocyte Rectifier Potassium Channel Scorpion Toxin Agar Bridge Thallium Concentration
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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