Imaging and Quantification of Recycled KATP Channels
This chapter describes immunochemistry-based methods to investigate recycling of membrane proteins at the cell surface. Two methods are described, one qualitative and the other quantitative. Both methods consist of two rounds of extracellular antibody cap
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Introduction Recycling membrane proteins are internalized from the plasma membrane into early and sorting endosomes from where they can either return to the cell surface via a Rab4-dependent pathway (1) or enter the endosomal recycling compartment (ERC) from where their return to the plasma membrane occurs via Rab11-dependent pathways (2). A limited number of membrane proteins use a somewhat convoluted pathway, involving trafficking from the ERC to the cell surface via the trans-Golgi network (3). Trafficking mechanisms for recycling are extensively reviewed by Maxfield and McGraw (4) and Grant and Donaldson (5). Recycling of internalized membrane proteins back to the cell surface has been reported for a range of membrane proteins including ion channels, receptors, and transporters. It represents an efficient mechanism by which prompt delivery of proteins to the plasma membrane occurs in response to changes in cell physiology. For example, recycling endosomes supply APMA receptor for long-term potentiation in neurons (6), b-adrenergic receptors for
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_18, © Springer Science+Business Media, LLC 2013
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Christopher J. Cockcroft
receptor resensitization (7), and GLUT-4 transporters for glucose uptake by adipose and muscle (8). This chapter describes methods for imaging and quantification of recycled channels. Using the KATP channel as an example, we describe immunochemistry-based methods for investigating membrane protein recycling (9). These methods employ an antibody capture approach that requires an antibody that recognizes an extracellular epitope (native or engineered) of the membrane protein of interest. A similar approach has also been described for investigating endocytosis of KATP channels (10, 11). In the examples presented here, we also use an HA-epitope (YPYDVPDYA) engineered into the extracellular loop of the Kir6.2 subunit of the KATP channel (12). The HA-tagged Kir6.2 was co-expressed with the sulphonylurea receptor 1(SUR1) in HEK-293 cells to produce HA-tagged KATP channels (KATP-HA) at the cell surface (under normal conditions neither subunit is capable of independent surface expression). Internalized primary antibody-channel complexes can recycle back to the cell surface to capture the probe-conjugated secondary antibodies (see Note 1). For example, recycled channels can be detected by using either confocal fluorescent microscopy or image-captured fluorescent-conjugated secondary antibodies (Fig. 1); following these steps, cells can then be fixed and permeabilized to stain either non-recycled channels or intracellular organelle markers. Alternatively, recycling can be quantified using chemiluminescence to assay-captured HRP-conjugated secondary antibody by assaying enzyme activity (Fig. 2).
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Materials
2.1 Preparation of Coverslips and Cells
1. No. 7 curved watchmaker forceps. 2. Borosilicate glass coverslips (13 mm). 3. HEK293 cell line (ECACC). 4. Dulbecco’s
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