Surface Expression of Membrane Proteins Detected Using an ELISA-based Luminometric Assay

Undergraduate #124
Discipline: Biological Sciences
Subcategory: Physiology and Health

Maria Montes - Garden City Community College
Co-Author(s): Kristopher Silver, James Lillich, and Peying Fong, Kansas State University

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Solutes move across cell membranes through transporters and channels, which must be synthesized and moved to their appropriate site(s) of action. Cell signaling mechanisms, which can be modulated by pharmacological agents, affect trafficking of proteins, including transport molecules. The present study examines membrane distribution of two different transport proteins, a sodium-coupled transporter (SLC5A8) and a voltagegated potassium channel (Kv1.4), using cell surface luminometry (Zerangue and Schwappach, 1999). Functionality of the assay was tested by transfecting human embryonic kidney (HEK) 293 cells with constructs encoding human SLC5A8 bearing either an extracellular HA tag (test) or, alternatively, an intracellular V5 tag (control). Control and test cells were stained with HRPlabeled rat monoclonal anti-HA antibody and surface luminometry performed after adding peroxidase substrate. Cells expressing the HA-hSLC5A8 produced higher luminescence than those expressing the V5 epitope, thereby validating the assay methodology. The voltage-gated potassium channel, Kv1.4, is necessary for cell migration and restitution after damage to the intestinal epithelium. Importantly, non-steroidal antiinflammatory drugs (NSAIDs) impede restitution (Freeman et al., 2007). To test whether NSAIDs exert effects at the level of Kv1.4 channel trafficking, intestinal epithelial cells (IEC6) transfected with extracellularly myc-tagged Kv1.4 were treated with NSAIDS (either indomethacin, NS-398 or SC-560) or control vehicle (0.1% DMSO) and stained with anti-myc antibody. Our initial experiments show decreased Kv1.4 surface expression in NSAIDtreated IEC6 cells. In conclusion, luminometric quantification of surface protein expression can offer insights into manifold cellular processes, including mechanisms of ion channelmediated epithelial restitution.

Funder Acknowledgement(s): NSF Grant 1305059, National Institutes of Health / National Institute of General Medical Sciences Award 1R15GM101674-10A1 (to PF)

Faculty Advisor: Arthur Nonhof,