Function recovery after chemobleaching (FRAC): evidence for activity silent membrane receptors on cell surface |
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| Authors: | Sun Haiyan Shikano Sojin Xiong Qiaojie Li Min |
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| Affiliation: | Department of Neuroscience and High Throughput Biology Center, The Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA. |
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| Abstract: | Membrane proteins represent approximately 30% of the proteome of both prokaryotes and eukaryotes. Unique to cell surface receptors is their biogenesis pathway, which involves vesicular trafficking from the endoplasmic reticulum through the Golgi apparatus and to the cell surface. Increasing evidence suggests specific regulation of biogenesis for different membrane receptors, hence affecting their surface expression. We report the development of a pulse-chase assay to monitor function recovery after chemobleaching (FRAC) to probe the transit time of the Kir2.1 K+ channel to reach the cell surface. Our results reveal that the channel activity is contributed by a small fraction of channel protein, providing evidence of activity-silent "sleeping" molecules on the cell surface. This method distinguishes molecular density from functional density, and the assay strategy is generally applicable to other membrane receptors. The ability of the reported method to access the biogenesis pathways in a high-throughput manner facilitates the identification and evaluation of molecules affecting receptor trafficking. |
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| Keywords: | covalent modification fluorescence recovery after photobleaching ion channels signaling trafficking |
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