Using Total Internal Reflection Fluorescence Microscopy to Observe Ion Channel Trafficking and Assembly
Ion channels are integral membrane proteins that allow the flow of ions across membranes down their electrochemical gradients and are a major determinant of cellular excitability. They play an important role in a variety of biological processes as diverse
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Introduction Membranes consist of a lipid bilayer which makes them impermeable for most molecules including ions. In biological membranes, ion channels form pores which, with varying selectivity, allow ions to pass at a high rate through the membrane down their electrochemical gradient. Ion channel activity can be modulated directly by ligand binding, membrane potential, second messengers (such as Ca2+ or cyclic-AMP), protein–protein interactions, and phosphorylation (1). Potassium channels are the largest family of ion channels and they are important for maintenance of the membrane resting potential, hormone secretion, regulation of excitability, repolarization of the action potential, and its shape and frequency (2–4). Potassium channels are homo- or heteromeric proteins consisting of the poreforming alpha subunit and in some cases additional beta subunits, which can be cytosolic or integral membrane proteins (5).
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_15, © Springer Science+Business Media, LLC 2013
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Sarah Schwarzer et al.
The function of ion channels can be studied in exquisite detail using patch clamping to the point where opening of single channels can be observed with a millisecond time resolution. In addition to this measurement of electrical activity it would be revealing to directly observe single vesicles carrying ion channels towards the plasma membrane and the movement of single ion channels in the membrane of cells. Confocal microscopy allows the analysis of the ion channel subcellular localization and can identify single vesicles; however it is not easy to identify single molecules. In contrast, laser-based total internal reflection fluorescence microscopy (TIRFM) combined with use of highly sensitive camera systems has sufficient signal-to-noise ratio and sensitivity to identify single fluorophores. Furthermore, by tracking the motion of individual molecules moving at the plasma membrane, using automated algorithms, information about protein mobility, membrane structure, and protein–membrane and protein–protein interaction can be obtained. We have developed methodologies to study the trafficking, membrane fusion, and single molecule diffusion of potassium channels in the plasma membrane of living cells using a combination of recombinant gene technology, cell transfection, and novel imaging techniques (6). We first tag the protein of interest with a fluorescent protein such as enhanced green fluorescent protein (eGFP) or monomeric red fluorescent protein (mRFP) using standard cloning methodologies. Transfection techniques then allow the expression of a low level of the channel in HEK293 or the cardiac derived HL-1 cells (7). Using tagged fluorescent proteins is convenient, because we know they are bound to the protein of interest; the biogenesis and transport of the channel can be studied and adjusting the transfection method can regulate the expression level.
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Materials Cloning
1. pEGFP-N1
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