Combinatorial cysteine mutagenesis reveals a critical intramonomer role for cysteines in prestin voltage sensing.

Biophysical Journal
Jun-Ping BaiDhasakumar Navaratnam

Abstract

Prestin is a member of the SLC26 family of anion transporters and is responsible for electromotility in outer hair cells, the basis of cochlear amplification in mammals. It is an anion transporting transmembrane protein, possessing nine cysteine residues, which generates voltage-dependent charge movement. We determine the role these cysteine residues play in the voltage sensing capabilities of prestin. Mutations of any single cysteine residue had little or no effect on charge movement. However, using combinatorial substitution mutants, we identified a cysteine residue pair (C415 and either C192 or C196) whose mutation reduced or eliminated charge movement. Furthermore, we show biochemically that surface expression of mutants with markedly reduced functionality can be near normal; however, we identify two monomers of the protein on the surface of the cell, the larger of which correlates with surface charge movement. Because we showed previously by Förster resonance energy transfer that monomer interactions are required for charge movement, we tested whether disulfide interactions were required for dimerization. Using Western blots to detect oligomerization of the protein in which variable numbers of cysteines up to and including...Continue Reading

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Citations

Feb 9, 2016·Hearing Research·Channy ParkFederico Kalinec
Dec 24, 2013·Hearing Research·David Z Z HeKirk W Beisel
Oct 8, 2009·Pflügers Archiv : European journal of physiology·Ghanshyam P SinhaKuni H Iwasa

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