Abstract
A novel truncate of Influenza A M2 protein (residues 22-62), incorporated into a uniquely tailored proteoliposome proton uptake assay, demonstrated proton flux more characteristic of an ion transporter than a traditional ion "channel." The liposome paradigm was essential for testing the conductance activity of this M2 truncate at a range of extraphysiological pHs appropriate for channel vs. transport function determination. In addition to transporter-typical proton flux, M2(22-62) showed the key characteristics of functional integrity: selective proton uptake into liposomes and block of uptake by amantadine. Two sets of proteoliposome proton flux assays were carried out, Set 1 at pH values of 6.5, 6.0. 5.5, 5.0, and 4.5; Set 2 at pH values of 6.25, 6.0, 5.75, 5.5, 5.25, 5.0, and 4.75. Observed flux rates followed a proton transport saturation curve similar to that observed in mouse erythroleukemia cells1. Proton transport was maximal at pH 5.5 in Set 1 (139 H+/second/tetramer) and at pH 5.75 in Set 2 (43 H+/second/tetramer). Amantadine block was strongest at pH 5.5 in Set 1 and 6.25 in Set 2, and apparent desensitization of the protein severely reduced proton flux and amantadine sensitivity below pH 5.5 in both sets of experiments. Decreased external pH increased proton uptake with an apparent pKa of 6 (Set 1) or 6.5 (Set 2). These data indicate acid activation of M2(22-62) between pH 5.5-6, optimal amantadine block between pH 5.5-6.25, and a loss of peptide functionality between pH 5.9-4.7.
Degree
MS
College and Department
Life Sciences; Physiology and Developmental Biology
Rights
http://lib.byu.edu/about/copyright/
BYU ScholarsArchive Citation
Peterson, Emily, "Proteoliposome Proton Flux Assays Establish Net Conductance, pH-Sensitivity, and Functional Integrity of a Novel Truncate of the M2 Ion "Channel" of Influenza A" (2010). Theses and Dissertations. 2420.
https://scholarsarchive.byu.edu/etd/2420
Date Submitted
2010-11-19
Document Type
Thesis
Handle
http://hdl.lib.byu.edu/1877/etd4057
Keywords
Influenza A, M2 protein, proton transporter, proton channel, acid activation, proteoliposome, liposome, amantadine
Language
English