Proteoliposome Proton Flux Assays Establish Net Conductance, pH-Sensitivity, and Functional Integrity of a Novel Truncate of the M2 Ion "Channel" of Influenza A
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 co...
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| Format: | Others |
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BYU ScholarsArchive
2010
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| Online Access: | https://scholarsarchive.byu.edu/etd/2420 https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=3419&context=etd |
| Summary: | 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. |
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