Cysteine Mutagenesis Reveals Transmembrane Residues Associated with Charge Translocation in Prestin

Abstract

The solute carrier transmembrane protein prestin (SLC26A5) drives an active electromechanical transduction process in cochlear outer hair cells that increases hearing sensitivity and frequency discrimination in mammals. A large intramembraneous charge movement, the nonlinear capacitance (NLC), is the electrical signature of prestin function. The transmembrane domain (TMD) helices and residues involved in the intramembrane charge displacement remain unknown. We have performed cysteine-scanning mutagenesis with serine or valine replacement to investigate the importance of cysteine residues to prestin structure and function. The distribution of oligomeric states and membrane abundance of prestin was also probed to investigate whether cysteine residues participate in prestin oligomerization and/or NLC. Our results reveal that 1) Cys-196 (TMD 4) and Cys-415 (TMD 10) do not tolerate serine replacement, and thus maintaining hydrophobicity at these locations is important for the mechanism of charge movement; 2) Cys-260 (TMD 6) and Cys-381 (TMD 9) tolerate serine replacement and are probably water-exposed; and 3) if disulfide bonds are present, they do not serve a functional role as measured via NLC. These novel findings are consistent with a recent structural model, which proposes that prestin contains an occluded aqueous pore, and we posit that the orientations of transmembrane domain helices 4 and 10 are essential for proper prestin function.Prestin is an essential component of the voltage-sensitive molecular motor of outer hair cells that converts changes in transmembrane potential into mechanical forces. This transduction causes outer hair cells to undergo a characteristic length change termed electromotility that enhances sound amplification and frequency selectivity (1, 2). In the absence of prestin, hearing thresholds are effectively elevated 40 -60 dB in mice (3). A large displacement current, analogous to the gating current in ion channels, has been established as a reliable electrical signature for electromotility (4,5). This electrical signature is referred to as the nonlinear capacitance (NLC) 4 and requires the presence of intracellular chloride (6).Very little is known about the structure of prestin and its relationship to prestin function. Several topologies have been proposed based on hydrophobicity and sequence conservation between prestin and the family of solute anion transporters to which it belongs. Proteins of the solute carrier family 26 (SLC26) probably span the membrane 10 -14 times (7-9). Because prestin (SLC26A5) has been shown to have intracellular N and C termini (10), topologies have been proposed having an even number of transmembrane domains. Ten-pass and reentrant loop 12-pass models of prestin secondary structure have been proposed (Fig. 1 and supplemental Table S1) and refined based on knowledge of phosphorylation sites (11), evolutionary analysis (12), and conflicting reports of N-linked glycosylation (13, 14), a result possibly due to expression in different model cell systems. Cur...

The roles of conserved and nonconserved cysteinyl residues in the oligomerization and function of mammalian prestin

“…The percentage FRET obtained with pgPT/pgPY, at 7%, is substantial and comparable to recent observations by us and other laboratories (McGuire et al 2010;Navaratnam et al 2005;Wu et al 2007). The higher FRET percentage of the pLink construct (20%) is likely a consequence of the forced 1:1 donor-acceptor ratio and the short distance between the donor and acceptor fluorophores in the construct.…”

“…We do not find the apparent hyperpolarized shift in V pk in wild-type prestin relative to isolated hair cells (Bai et al 2010;McGuire et al 2010). Our average V pk value for wild-type prestin, Ϫ51 mV, is much closer to reported V pk values for OHCs and prestin-transfected HEK cells.…”

“…We have chosen not to report Q/C l results as a measure of membrane incorporation, as others have done (Bai et al 2010;McGuire et al 2010), because they do not have the significance ascribed to them. In transfected cell experiments, the cells selected for examination are a vanishingly small subset of those available to the experimenter and are undoubtedly selected to be those with the clearest membrane label.…”

Membrane prestin expression correlates with the magnitude of prestin-associated charge movement

“…Our recording techniques are fully described earlier (McGuire et al, 2010; Rajagopalan et al, 2006), but a brief description follows. Murine OHCs were used within 4 hours of isolation and within 1 hour of being in the recording chamber at RT.…”

“…The conductance was also determined experimentally with a DC protocol, as described earlier (Rajagopalan et al, 2006). Capacitance curves were fit to a Boltzmann equation, from which charge density (Q max /C lin ), voltage at peak capacitance (V pkc ), and the unitary change valence or maximum slope of the Boltzmann fit ( z ) were determined as previously described (Bian et al, 2010; McGuire et al, 2010; Oliver and Fakler, 1999; Rajagopalan et al, 2006). …”