Visualizing ion channel dynamics at the plasma membrane.
Cardiac ion channels are surprisingly dynamic in nature, and are continuously formed, trafficked to specific subregions of plasma membrane, inserted in the plasma membrane, and removed to be degraded or recycled. Because of these movements, which affect channel availability, ion channel function is dependent on not just channel biophysical properties but channel trafficking as well. The development of molecular techniques to tag proteins of interest with fluorescent and other genetically encoded proteins, and of advanced imaging modalities such as total internal reflection microscopy (TIRF), have created new opportunities to understand the intracellular movement of proteins near the plasma membrane and their dynamics therein. In this article we present approaches for ion channel biologists to the use of fluorescent and nonfluorescent fusion proteins, techniques for cloning and expression of fusion proteins in mammalian cells, and imaging techniques for live-cell high-resolution microscopy. For illustration, original data are presented on creation of a stable cell line capable of inducible expression of connexin 43 tagged to green fluorescent protein and its distribution viewed with both wide-field epifluorescence and TIRF microscopy. With revolutionary advances in fluorescence microscopy, ion channel biologists are now entering a new realm of studying channel function, which is to understand the mechanisms of channel trafficking.
Smyth
James W
JW
Cardiovascular Research Institute and Department of Medicine, University of California San Francisco, San Francisco, California, USA.
Shaw
Robin M
RM
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D006801:Humans; D007473:Ion Channels / Q000378:metabolism / Q000648:ultrastructure*; D017136:Ion Transport / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods*
D007473:Ion Channels