Prestin in HEK cells is an obligate tetramer
| Autor: | Michael G. Nichols, Richard Hallworth |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
biology
Physiology Chemistry General Neuroscience Anion Transport Proteins Cell Membrane HEK 293 cells Articles Transport protein Green fluorescent protein Cell membrane Motor protein HEK293 Cells medicine.anatomical_structure Förster resonance energy transfer Tetramer Sulfate Transporters medicine biology.protein Biophysics Humans sense organs Prestin Dimerization |
| Zdroj: | Journal of Neurophysiology. 107:5-11 |
| ISSN: | 1522-1598 0022-3077 |
| DOI: | 10.1152/jn.00728.2011 |
| Popis: | The unusual membrane motor protein prestin is essential for mammalian hearing and for the survival of cochlear outer hair cells. While prestin has been demonstrated to be a homooligomer, by Western blot and FRET analyses, the stoichiometry of self association is unclear. Prestin, coupled to the enhanced green fluorescent protein, was synthesized and membrane targeted in human embryonic kidney cells by plasmid transfection. Fragments of membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent in size with single molecules were observed. Under continuous excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background levels was 2.7. A binomial model of prestin oligomerization indicated that prestin was most likely a tetramer, and that a fraction of the green fluorescent protein molecules was dark. As a positive control, the same procedure was applied to cells transfected with plasmids coding for the human cyclic nucleotide-gated ion channel A3 subunit (again coupled to the enhanced green fluorescent protein), which is an obligate tetramer. The average step count for this molecule was also 2.7. This result implies that in cell membranes prestin oligomerizes to a tetramer. |
| Databáze: | OpenAIRE |
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