SLC26A transporters are 11–13 transmembrane antiporters that promote the movement of anionic substrates (chloride, iodide, bicarbonate, and formate) with different specificities (Markovich, 2001
; Vincourt et al., 2003
; Mount and Romero, 2004
). Mammalian prestins are unique because they have not been conclusively associated with conventional transport capabilities, although a recent model hypothesizes an antiport function (Muallem and Ashmore, 2006
). The presence of ~12 TMs and the ability to couple anion exchange to the chemiosmotic gradient indicates common features between the human SLC26A family (transport classification number TC 2.A.53) and the major facilitator superfamily (MFS) (TC number 2.A.1; transporter classification database, http://www.tcdb.org
). The transport mechanism of MFS members are postulated to involve alternating accessibility of a central substrate-binding site to either surface of the membrane (Mitchell, 1957
) caused by a conformational change in the transporter triggered by anion binding. A similar mechanism could explain conformational changes in prestin, triggered by cell potential changes.
Previous prestin functional mutation studies examined nonconserved charged residues (Oliver et al., 2001
), glycosylation sites (Matsuda et al., 2004
), phosphorylation sites (Deak et al., 2005
), as well as domain swapping (Zheng et al., 2005
) or C-and N-terminal deletions (Navaratnam et al., 2005
; Zheng et al., 2005
) and provided insights into prestin function by identifying residues or regions that affect prestin NLC. We used ET to identify critical functional residues in prestin and conducted a systematic mutational analysis, changing size, charge, or polarity of selected residues to better understand their contribution to prestin function.
ET analysis of prestin homologs revealed two highly ranked residue clusters in the region of the sulfate anion transporter motif (residues 109–130) (Mount and Romero, 2004
), based on our predicted topology map of prestin (). Replacement of all residues in each cluster with tryptophan eliminated NLC (). Additional systematic replacements of cluster 1 residues (A100, A102, and L104) revealed size-dependent sensitivity to residue substitutions (). Relatively normal NLC profiles were only seen when these residues were substituted with a smaller or isostructural amino acid. Cluster 2 residues (L113 and F117) did not appear to be dependent on residue size; replacement of L113 with W resulted in a bell-shaped, albeit peak-shifted NLC, whereas F117W closely resembled WT (), but smaller substitutions may yield additional information on the role of this residue.
Interestingly, effects on NLC were observed only when residue size was changed, with hydrophobicity not a major contributing factor (except in the case of A102). In addition, these residues are not charged, leading to the assumption that they are not directly involved in charge movement. This is corroborated by the fact that certain substitutions at these residues retain NLC, albeit with altered peak characteristics. The identified residues, therefore, may be involved in helix packing, and perhaps in allosteric interactions, that influence conformational changes associated with charge movement.
A double mutant containing a smaller substitution (A102G) on helix 1 and a larger substitution (L113W) on helix 2 behaves similar to the L113W single mutant (). A102 and L113 are adjacent to one another in helices 1 and 2, respectively; therefore, effects of a larger substitution at one of these residues might be compensated by a smaller substitution at the other. The Vpkc of the double mutant is slightly shifted toward the WT value (from the L113W value), but this is not statistically significant. The lack of compensation may be attributable to these two residues being not precisely in register or to the L113W substitution being too bulky for the smaller A102G substitution to compensate. This result, however, affirms that larger substitutions dominate the effect on prestin NLC, corroborating the theory of packing between residues.
All mutants that exhibited NLC showed charge densities comparable with WT, within SD limits (). The double mutant showed significantly lowered charge density, perhaps attributable to structural perturbation from multiple substitutions.
Figure 6 NLC characteristics of prestin and mutants. Average charge density (A) and average voltage at peak capacitance Vpkc (B) of single mutants are compared with wild type. Mutants A100V, A100L, A100W, A102V, A102L, A102W, and L104W are not represented in (more ...)
The A102S substitution showed a depolarizing shift in Vpkc, although the corresponding substitution at A100 showed WT-like NLC. Serine is more hydrophilic than alanine and might have the effect of perturbing protein–lipid interactions. Although A100 is tolerant of this change, the serine substitution at A102 on the opposite face of the helix does exhibit a significant effect on NLC. This might be an indication that the helical face containing A102 makes more contact with membrane components and/or with hydrophobic protein surfaces in the transmembrane region and therefore is less tolerant of a hydrophilic substitution.
An alanine outside the identified ET clusters was chosen to test the accuracy of ET predictions. A305 is located at the extracellular membrane interface of helix 7, at a similar position as A100, A102, and/or L104 in helix 1 of our topology map. Substitution of this residue with valine had no effect on the NLC profile (), indicating that perturbation of helix packing in this region does not affect NLC.
We have therefore identified a region in prestin that is critical for normal prestin activity. This region, located near the sulfate anion transporter motif, appears to be tightly packed, intolerant of alterations in residue bulk, and may be involved directly or allosterically in conformational changes during prestin activity. It is worth mentioning that mutational studies on a plant sulfate transporter SHST1 indicate that tight packing of residues in helices 1 and 2 is necessary for sulfate transport by this protein (Shelden et al., 2001
). It remains to be seen whether this region is directly involved in anion interactions that are essential for prestin activity (Oliver et al., 2001
; Rybalchenko and Santos-Sacchi, 2003
It has been proposed that intracellular chloride ions function as the voltage sensor of prestin, binding to a site in prestin, and translocating within the membrane in response to transmembrane voltage changes (Oliver et al., 2001
). However, an anion-binding site on prestin, or any SLC26A transporter, has not been identified. Recent analyses of chloride-binding mechanisms of transporters and channels indicate that chloride is coordinated chiefly by partial positive charges from main-chain amides in these proteins (Dutzler et al., 2002
; MacKinnon, 2004
), with selectivity conferred by the size of the filter and not charge. By these criteria, helices 1 and 2 of prestin do indeed appear to be equipped to form a chloride-binding pocket; additional mutational studies are necessary for more detailed analysis.
Whether or not helices 1 and 2 line the actual chloride-binding pocket of prestin, it is clear that perturbations in the packing of these helices affect activity. In fact, the data can be correlated in broad mechanistic terms with the predicted topology map of prestin. A100 and A102 are two residues apart and therefore would be positioned on opposing surfaces of helix 1. Therefore, replacing A100 with a smaller side chain would have an opposite effect from a similar replacement at A102, in terms of helix packing. Although the direct effect on helix packing is unknown, the effect is very noticeable in terms of nonlinear capacitance. Replacement of A100 and A102 with the smaller glycine side chain shifts Vpkc of prestin NLC in opposite directions, suggesting that the packing of helix 1 against its adjacent helices is altered in opposing ways, which can be quantitatively correlated to changes in NLC characteristics. Furthermore, if helices 1 and 2 pack against each other, based on our two-dimensional topology, then replacement of L113 with W would have the effect of moving these helices apart by introducing steric bulk between them, whereas replacement of A100 with G would create more freedom for helix 1, allowing it to pack less closely with helix 2. In other words, L113W and A100G would produce similar effects, and, in fact, the data show that the effect of both these mutants is a positive shift of Vpkc, albeit to different extents. In addition, the A102G/L113W double mutant is not statistically distinguishable from L113W, indicating the dominance of a larger over a smaller substitution, as expected if the residues are closely packed.
A variety of manipulations that result in Vpkc
changes include changes in membrane properties induced mechanically (Kakehata and Santos-Sacchi, 1995
; Santos-Sacchi et al., 2001
; Dong and Iwasa, 2004
), chemically (Santos-Sacchi, 1991
; Shehata et al., 1991
; Tunstall et al., 1995
; Kakehata and Santos-Sacchi, 1996
; Lue et al., 2001
), by temperature (Meltzer and Santos-Sacchi, 2001
), or mutations (Oliver et al., 2001
; Matsuda et al., 2004
; Deak et al., 2005
; Navaratnam et al., 2005
; Zheng et al., 2005
). Electromotility in OHCs represents a strong, reciprocal coupling of electrical and mechanical energy; therefore, changes in stress/polarization of the membrane result in peak shifts. We suggest that point mutations in the sulfate transporter motif alter the integrity of the membrane domains around prestin such that changes in both the electrical field in the membrane or membrane tension result in the observed peak shifts.
In summary, we used ET analysis to identify candidate functionally important residues in prestin. We generated systematic mutations at each residue, varying hydrophobicity, polarity, and size. The results indicate a possible functional role for the region of the conserved SLC26A sulfate anion transporter motif in prestin activity. Mutations of the corresponding residues in prestin orthologs such as pendrin would yield information of the functional significance, if any, of this region in transport processes of other SLC26A family proteins. In addition, substitutions of these amino acids in other SLC26A proteins might be used to confer prestin-like activity to these proteins.