Essential Helix Interactions in the Anion Transporter Domain of Prestin Revealed by Evolutionary Trace Analysis

Rajagopalan, Lavanya; Patel, Nimish; Madabushi, Srinivasan; Goddard, Julie Anne; Anjan, Venkat; Li, Feng; Shope, Cindy; Farrell, Brenda; Lichtarge, Olivier; Davidson, Amy L.; Brownell, William E.; Pereira, Fred A.

doi:10.1523/jneurosci.2734-06.2006

Cited by 50 publications

(61 citation statements)

References 49 publications

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“…The charge density was 4,432±1,132 e − /μm 2 . This was similar to charge densities within HEK cells transfected with prestin (range: 3,700-5,500; Rajagopalan et al 2006;Zheng et al 2000). These data indicate that the HDAd-prestin-GFP virus was able to transduce cells and lead to the expression of functional prestin protein.…”

Section: Ha-prestin Expression and Nonlinear Capacitance Of Hek Cellssupporting

confidence: 70%

“…The prestin cDNA was from the gerbil. The pCMV-HAprestin-IRES-hrGFP-1a mammalian expression plasmid independently expresses hemagglutinin (HA)-tagged prestin and GFP, which allows for easy identification of transduced cells (Rajagopalan et al 2006;Sturm et al 2007). The resultant vector was linearized with Mlu1 and inserted into the compatible Asc I restriction site in the HDAd vector (pΔ25.3E4 vector), which was derived from pΔ28E4 by deletion of the 3 kb Nhe I fragment (Toietta et al 2002).…”

Section: Creation Of Plasmidsmentioning

confidence: 99%

“…The electrical signature of prestin function is a nonlinear capacitance, which is thought to reflect anion movement associated with changes in prestin conformation (He et al 2006;Oliver et al 2001). Prestin transfection of human embryonic kidney (HEK) cells results in a nonlinear capacitance (Deak et al 2005;Rajagopalan et al 2006;Santos-Sacchi 2002;Zheng et al 2000). In adult OHCs, electromotility and nonlinear capacitance are highly correlated (Ashmore 1990;Dallos and Fakler 2002;Santos-Sacchi 1991a).…”

Section: Introductionmentioning

confidence: 99%

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Functional Prestin Transduction of Immature Outer Hair Cells from Normal and Prestin-Null Mice

Xia

Wooltorton

Palmer

et al. 2008

JARO

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Prestin is a membrane protein in the outer hair cell (OHC) that has been shown to be essential for electromotility. OHCs from prestin-null mice do not express prestin, do not have a nonlinear capacitance (the electrical signature of electromotility), and are smaller in size than wild-type OHCs. We sought to determine whether prestin-null OHCs can be transduced to incorporate functional prestin protein in a normal fashion. A recombinant helper-dependent adenovirus expressing prestin and green fluorescent protein (HDAd-prestin-GFP) was created and tested in human embryonic kidney cells (HEK cells). Transduced HEK cells demonstrated membrane expression of prestin and nonlinear capacitance. HDAd-prestin-GFP was then applied to cochlear sensory epithelium explants harvested from wild-type and prestin-null mice at postnatal days 2-3, the age at which native prestin is just beginning to become functional in wildtype mice. At postnatal days 4-5, we investigated transduced OHCs for (1) their prestin expression pattern as revealed by immunofluorescence; (2) their cell surface area as measured by linear capacitance; and (3) their prestin function as indicated by nonlinear capacitance. HDAd-prestin-GFP efficiently transduced OHCs of both genotypes and prestin protein localized to the plasma membrane. Whole-cell voltage clamp studies revealed a nonlinear capacitance in transduced wild-type and prestin-null OHCs, but not in non-transduced cells of either genotype. Prestin transduction did not increase the linear capacitance (cell surface area) for either genotype. In peak nonlinear capacitance, voltage at peak nonlinear capacitance, charge density of the nonlinear capacitance, and shape of the voltage-capacitance curves, the transduced cells of the two genotypes resembled each other and previously reported data from adult wildtype mouse OHCs. Thus, prestin introduced into prestin-deficient OHCs segregates normally to the cell membrane and generates a normal nonlinear capacitance, indicative of normal prestin function.

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Section: Ha-prestin Expression and Nonlinear Capacitance Of Hek Cellssupporting

confidence: 70%

Section: Creation Of Plasmidsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Functional Prestin Transduction of Immature Outer Hair Cells from Normal and Prestin-Null Mice

Xia

Wooltorton

Palmer

et al. 2008

JARO

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“…The newly derived functions are typically associated with regions of a protein that have high peptide sequence variability (Kimura, 1983). Evolutionary studies combined with comparative genomic and bioinformatic analyses have identified two regions of amino acid sequences that are highly invariant among eutherian species, but highly polymorphic among non-mammalian vertebrates and SLC26A paralogs (Franchini and Elgoyhen, 2006;Rajagopalan et al, 2006;Okoruwa et al, 2008;Elgoyhen and Franchini, 2011). Based on the fact that motor capability is an innovation of mammalian prestin, we predict that the structural innovation underlying motor function in eutherian prestin evolved from a site within the SulpTP domain that has well-conserved amino acid composition among mammalian prestins but is highly variable in non-mammalian prestin orthologs.…”

Section: Introductionmentioning

confidence: 99%

A motif of eleven amino acids is a structural adaptation that facilitates motor capability of eutherian prestin

Tan

Pecka

Tang

et al. 2012

Journal of Cell Science

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SummaryCochlear outer hair cells (OHCs) alter their length in response to transmembrane voltage changes. This so-called electromotility is the result of conformational changes of membrane-bound prestin. Prestin-based OHC motility is thought to be responsible for cochlear amplification, which contributes to the exquisite frequency selectivity and sensitivity of mammalian hearing. Prestin belongs to an anion transporter family, the solute carrier protein 26A (SLC26A). Prestin is unique in this family in that it functions as a voltage-dependent motor protein manifested by two hallmarks, nonlinear capacitance and motility. Evidence suggests that prestin orthologs from zebrafish and chicken are anion exchangers or transporters with no motor function. We identified a segment of 11 amino acid residues in eutherian prestin that is extremely conserved among eutherian species but highly variable among non-mammalian orthologs and SLC26A paralogs. To determine whether this sequence represents a motif that facilitates motor function in eutherian prestin, we utilized a chimeric approach by swapping corresponding residues from the zebrafish and chicken with those of gerbil. Motility and nonlinear capacitance were measured from chimeric prestin-transfected human embryonic kidney 293 cells using a voltage-clamp technique and photodiode-based displacement measurement system. We observed a gain of motor function with both of the hallmarks in the chimeric prestin without loss of transport function. Our results show, for the first time, that the substitution of a span of 11 amino acid residues confers the electrogenic anion transporters of zebrafish and chicken prestins with motor-like function. Thus, this motif represents the structural adaptation that assists gain of motor function in eutherian prestin.

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“…33,34 Case studies further showed that bona fide ET-guided mutants could then block, separate and even swap functions in vitro and in vivo. [35][36][37][38] ET thus predicts key functional determinants and enables their rational perturbation.…”

Section: Introductionmentioning

confidence: 99%

Sequence and structure continuity of evolutionary importance improves protein functional site discovery and annotation

et al. 2010

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Protein functional sites control most biological processes and are important targets for drug design and protein engineering. To characterize them, the evolutionary trace (ET) ranks the relative importance of residues according to their evolutionary variations. Generally, top-ranked residues cluster spatially to define evolutionary hotspots that predict functional sites in structures. Here, various functions that measure the physical continuity of ET ranks among neighboring residues in the structure, or in the sequence, are shown to inform sequence selection and to improve functional site resolution. This is shown first, in 110 proteins, for which the overlap between top-ranked residues and actual functional sites rose by 8% in significance. Then, on a structural proteomic scale, optimized ET led to better 3D structure-function motifs (3D templates) and, in turn, to enzyme function prediction by the Evolutionary Trace Annotation (ETA) method with better sensitivity of (40% to 53%) and positive predictive value (93% to 94%). This suggests that the similarity of evolutionary importance among neighboring residues in the sequence and in the structure is a universal feature of protein evolution. In practice, this yields a tool for optimizing sequence selections for comparative analysis and, via ET, for better predictions of functional site and function. This should prove useful for the efficient mutational redesign of protein function and for pharmaceutical targeting.

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Essential Helix Interactions in the Anion Transporter Domain of Prestin Revealed by Evolutionary Trace Analysis

Cited by 50 publications

References 49 publications

Functional Prestin Transduction of Immature Outer Hair Cells from Normal and Prestin-Null Mice

Functional Prestin Transduction of Immature Outer Hair Cells from Normal and Prestin-Null Mice

A motif of eleven amino acids is a structural adaptation that facilitates motor capability of eutherian prestin

Sequence and structure continuity of evolutionary importance improves protein functional site discovery and annotation

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