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

Abstract

The creation of several prestin knockout and knockin mouse lines has demonstrated the importance of the intrinsic outer hair cell membrane protein prestin to mammalian hearing. However, the structure of prestin remains largely unknown, with even its major features in dispute. Several studies have suggested that prestin forms homo-oligomers that may be stabilized by disulfide bonds. Our phylogenetic analysis of prestin sequences across chordate classes suggested that the cysteinyl residues could be divided into three groups, depending on the extent of their conservation between prestin orthologs and paralogs or homologs. An alanine scan functional analysis was performed of all nine cysteinyl positions in mammalian prestin. Prestin function was assayed by measurement of prestin-associated nonlinear capacitance. Of the nine cysteine-alanine substitution mutations, all were properly membrane targeted and all demonstrated nonlinear capacitance. Four mutations (C124A, C192A, C260A, and C415A), all in nonconserved cysteinyl residues, significantly differed in their nonlinear capacitance properties compared with wild-type prestin. In the two most severely disrupted mutations, substitution of the polar residue seryl for cysteinyl restored normal function in one (C415S) but not the other (C124S). We assessed the relationship of prestin oligomerization to cysteine position using fluorescence resonance energy transfer. With one exception, cysteine-alanine substitutions did not significantly alter prestin-prestin interactions. The exception was C415A, one of the two nonconserved cysteinyl residues whose mutation to alanine caused the most disruption in function. We suggest that no disulfide bond is essential for prestin function. However, C415 likely participates by hydrogen bonding in both nonlinear capacitance and oligomerization.

Fig. 1.

Homology analysis of prestin. A: region, domain, and motif designation of the human prestin amino acid sequence: green, transmembrane region; blue, sulfate transporter (SulP) domain; red, sulfate transporter and anti-sigma factor antagonist (STAS) domain; and yellow, SulP motif. B: phylogeny of SulP family member proteins. The number at each node indicates the bootstrap number out of 100 repetitions. C: multiple sequence alignment of cysteinyl residues. Residue properties are indicated as follows: gray, small hydrophobic; green, medium-size hydrophobic; cyan, partial positive charge; blue, positive charge; orange, partial negative charge; red, negative charge; pink, proline; light purple, histidine; and yellow, special property.

Fig. 2.

Diagram showing the positions of the 9 cysteinyl residues in prestin, represented by small gray disks, in the 2 proposed membrane topologies (A, based on Deak et al. 2005; and B, based on Navaratnam et al. 2005).

Fig. 3.

Determination of successful membrane targeting of prestin mutations. A: typical human embryonic kidney (HEK) cell expressing prestin-enhanced green fluorescent protein (eGFP) (green) and labeled with WGA-633 (red). Inset: fluorescence intensity profiles of the line in the main image. B: average (+SE) of separation of WGA-633 and prestin in profiles, as in A, for wild-type (WT) and mutated prestins as indicated.

Fig. 4.

Normalized nonlinear capacitance (NLC) as a function of membrane potential for cysteine-alanine substitutions (solid squares) compared with wild-type prestin (open circles). Functions are not corrected for series resistance membrane potential error, which, although small (0.4% or less), means that the plots are shown for illustrative purposes only.

Fig. 5.

Summary of NLC properties of cysteine-alanine substitutions (solid squares) compared with wild-type prestin (open circles), plotted as mean + SE. A: peak membrane potential (V_pk). B: charge transfer (z). ☆P < 0.05; ★P < 0.001, different from WT.

Fig. 6.

A: normalized NLC of C124A (solid squares), C124S (shaded squares), and WT prestin (open circles) as a function of membrane potential. B: normalized NLC of C415A (solid squares), C415S (shaded squares), and WT prestin (open circles) as a function of membrane potential. Functions are not corrected for series resistance membrane potential error.

Fig. 7.

Averaged fluorescence resonance energy transfer (FRET) efficiencies of control and prestin construct-transfected cells. A: FRET efficiencies of cells transfected with negative controls (left) or positive controls (right) [ANOVA against negative control (3, 108, P < 0.01) and Student's t-tests]. *P < 0.05; ***P < 0.001. B: comparison of FRET efficiencies of positive (left) or negative control (dashed line) to WT/mutant donor/acceptor-cotransfected cells (right) [ANOVA against positive control (9, 320, P < 0.001), ANOVA against negative control (9, 295, P < 0.001), and Dunnett's 2-sided t-tests]. ***P < 0.001. Error bars indicate SE. See text for description of groups 1–3.

Fig. 8.

Total nonlinear charge transfer as a function of linear capacitance for 117 cells synthesizing WT prestin (slope = 0.0058 pC/pF, R = 0.118).