Combinatorial cysteine mutagenesis reveals a critical intramonomer role for cysteines in prestin voltage sensing.
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 all nine cysteine residues were mutated, we show that disulfide bond formation is not essential for dimer formation. Taken together, we believe these data indicate that intramembranous cysteines are constrained, possibly via disulfide bond formation, to ensure structural features of prestin required for normal voltage sensing and mechanical activity.
Bai
Jun-Ping
JP
Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, USA.
Surguchev
Alexei
A
Bian
Shumin
S
Song
Lei
L
Santos-Sacchi
Joseph
J
Navaratnam
Dhasakumar
D
D019943:Amino Acid Substitution; D000818:Animals; D027321:Anion Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D025802:Electric Capacitance; D004553:Electric Conductivity; D005849:Gerbillinae; D009154:Mutation; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
D027321:Anion Transport Proteins; D004220:Disulfides; D003545:Cysteine