The Structure-Function Relationship of Prestin: from an Evolutionary Perspective

dc.contributor.advisorHe, David Z. Z.en_US
dc.contributor.authorTan, Xiaodongen_US
dc.contributor.cuauthorTan, Xiaodongen_US
dc.date.accessioned2010-12-06T20:53:12Z
dc.date.available2011-05-15T07:45:04Z
dc.date.issued2010-10-14
dc.degree.committeeFritzsch, Bernden_US
dc.degree.committeeBeisel, Kirk W.en_US
dc.degree.committeeSoukup, Garrett A.en_US
dc.degree.committeeYee, John A.en_US
dc.degree.disciplineBiomedical Sciences (graduate program)en_US
dc.degree.grantorGraduate Schoolen_US
dc.degree.levelPhD (Doctor of Philosophy)en_US
dc.degree.namePh.D. in Biomedical Sciencesen_US
dc.description.abstractPrestin is the motor protein of cochlear outer hair cells. It belongs to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Members of this family serve two fundamentally distinct functions. While most members transport different anion substrates across a variety of epithelia, prestin (SLC26A5) uniquely functions as a voltage-dependent motor protein. This voltage-dependent response of prestin is accompanied by a charge movement, which is reflected in nonlinear capacitance (NLC). . Prestin is assumed to contain a tunnel-like structure which is only accessible from intracellular side, based on a hypothetical working model as a partial anion transporter proposed by Oliver et al. (2001). Intracellular anions such as Cl- act as the external voltage sensor of prestin and trigger the conformational change in the molecule, which in turn alters the surface area of plasma membrane. This study tends to gain insight on the functionally critical structures of prestin using comparative approach. Recent evidence suggests that prestin orthologs from zebrafish and chicken retain transporter function without motile capability. Mammalian prestin does not appear to transport anions across the cell membrane, while controversial studies suggest that mammalian prestin may also be able to transport anions and this transporter function is independent with its electromotility capability. These studies suggest that prestin is evolved from an anion transporter. In this study, I first examined the motor and transport functions of prestin and its orthologs from four different vertebrate species (zebrafish, chicken, platypus and gerbil), to gain insight regarding how these two physiological functions might have distinctly evolved. Somatic motility, voltage-dependent NLC and transporter function were measured in transfected human embryonic kidney (HEK) cells using voltage-clamp and anion uptake techniques. Zebrafish and chicken prestins both exhibit weak NLC with peaks significantly shifted in the depolarization (right) direction. This is contrasted by robust NLC with left-shifted peaks for both platypus and gerbil prestins. Platypus and gerbil prestins may only retain little transporter function in comparison with robust anion transport capacities in the zebrafish and chicken orthologs. Somatic motility is only detected in the platypus and gerbil prestins. There appears to be an inverse relationship between NLC and anion transport functions, whereas the motor function appears to have only emerged in mammalian prestin. Our results suggest that motor function is an innovation of mammalian prestin and is concurrent with diminished transporter capability. Evolutionary studies combined with comparative genomic and bioinformatic analyses have identified highly conserved sequences among mammalian prestins that show significant variability among nonmammalian vertebrate prestin orthologs and other SLC26A paralogs. Among these sequences is one segment of 11 residues. In the second part of this study, I investigated whether this region represents the minimal essential motif for the motor function. Chimeric proteins swapping corresponding residues of prestin orthologs from zebrafish and chicken with those from gerbil prestin (zebrafish prestin with gerbil sites, Zf(g), and chicken prestin with gerbil sites, Ck(g), respectively) were constructed. Motility, NLC and anion transport were examined. A gain of motor function was observed with two hallmarks (NLC and motility) in both Zf(g) and Ck(g) without loss of transport function. These results show that the substitution of only 11 amino acids is sufficient to confer motor function upon the electrogenic anion transporters of zebrafish and chicken prestins. Therefore, this segment represents the minimal essential motif for the motor. The regions or amino acids within prestin that are essential for voltage sensing are still unclear. Charged amino acids are likely to play an important role in voltage sensing because of their potential capability to serve as anion binding sites. Previous studies focused mostly on the property of charged side chains. In the third part of this study, the roles of three positively charged amino acids in voltage sensing of prestin were examined. I hypothesize that the size or charge location of the amino acid side chain is also an important factor for prestin function. Three positive amino acids in the putative tunnel region were selected based on molecular dynamics simulation and sequence alignment of prestin paralogs and orthologs, namely R197, K227 and K449. A series of substitutions using similarly charged amino acids (R to K and K to R) are constructed, assuming that R and K substitutions only affect size and charge orientation of the side chain. Negative (R / K to E), neutral (R / K to A) substitutions and combinations of substitutions at these three sites (double and triple mutations) were also constructed assuming that there are multiple anion binding sites in the molecule. The results show that all three sites are important for voltage sensing and that the size or charge orientation of the side chain is also a critical factor. Furthermore, negative correlations between the peak voltage of NLC or the total charge movement and the slope factor are observed, suggesting that other electrical features such as dielectric properties were changed by these substitutions rather than the number of charges or their traveling distance.en_US
dc.description.noteProQuest Traditional Publishing Optionen_US
dc.description.pagesxv, 92 pagesen_US
dc.embargo.terms2011-05-15
dc.identifier.otherTan-Dissertation.pdf
dc.identifier.urihttp://hdl.handle.net/10504/8734
dc.language.isoen_USen_US
dc.publisherCreighton Universityen_US
dc.publisher.locationOmaha, Nebraskaen_US
dc.rightsCopyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.en_US
dc.rights.holderXiaodong Tanen_US
dc.subject.meshBiological Evolutionen_US
dc.subject.meshAnion Transport Proteins--physiologyen_US
dc.subject.meshHair Cells, Auditory, Outer--physiologyen_US
dc.subject.meshMotor Activity--physiologyen_US
dc.titleThe Structure-Function Relationship of Prestin: from an Evolutionary Perspectiveen_US
dc.typeDissertation
Files
Original bundle
Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
Tan-Dissertation.pdf
Size:
4.5 MB
Format:
Adobe Portable Document Format
Description:
Xiaodong Tan, Dissertation (PDF)
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
1.61 KB
Format:
Item-specific license agreed upon to submission
Description: