PILOT PROJECT 8: CREATION OF A PENDRIN WITH BOTH MOTOR AND TRANSPORTER FUNCTIONS

试点项目 8:创建具有运动和转运功能的 PENDRIN

基本信息

  • 批准号:
    8360400
  • 负责人:
  • 金额:
    $ 4.19万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2011
  • 资助国家:
    美国
  • 起止时间:
    2011-07-01 至 2012-06-30
  • 项目状态:
    已结题

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A. Specific Aims The overall approach of this project is to observe the voltage sensitivity, electromotility and transporter functions of SLC26A proteins expressed on HEK 293 cells, and to determine the consequence induced by motif swapping mutations using the predicted domain crucial for motility. Specific aim 1: To determine whether voltage-dependency is a common feature of prestin's paralog in the SLC26A family. We hypothesize that SLC26A paralogs have some voltage dependency due to their structural similarities with prestin. We will measure nonlinear capacitance (NLC), which reflects the voltage-sensitivity, in HEK cells expressing SLC26A proteins. Transporter function will be measured by radioisotope-labeled anion uptake techniques. NLC will be measured under the whole-cell voltage-clamp. Specific aim 2: To determine whether prestin's paralog can gain motor function by adopting the electromotility (EM) motif from prestin. We hypothesize the domain swapping from gerbil prestin can convert the paralog into voltage-dependent motor. Mutagenesis techniques will be used to generate the chimera proteins. The NLC, motility and transporter functions of these chimeric paralogs will be examined using voltage-clamp, microchamber and radioisotope uptake techniques. B. Studies and Results Pendrin and prestin both belong to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. While pendrin (SLC26A4), found at the luminal membrane of follicular cells in the thyroid gland as well as in the endolymphatic duct and sac of the inner ear, is a chloride-iodide transporter, prestin (SLC26A5), expressed in the plasma membrane of cochlear outer hair cells, functions as a unique voltage-dependent motor. We recently identified a motif that is critical for the motor function of prestin. We questioned whether it was possible to create a chimeric pendrin protein with motor capability by adopting this motility motif from prestin. The chimeric pendrin was constructed by substituting residues 160-179 in human pendrin with residues 156-169 from gerbil prestin. Nonlinear capacitance and somatic motility, two hallmarks representing prestin function, were measured from chimeric pendrin-transfected human embryonic kidney 293 cells using voltage-clamp technique and photodiode-based displacement measurement system. We showed that a 14-amino-acid substitution from prestin was able to confer pendrin with voltage-dependent motor capability despite that pendrin has a different primary, secondary and tertiary structures. The molecular mechanism of motor function appeared to be the same as prestin, since the motor activity depended on intracellular chloride concentration and was blocked by salicylate treatment. Radioisotope-labeled formate uptake measurements showed that the modified pendrin retained the capability to transport formate, suggesting that the gain of motor function was not at the expense of its inherent transport capability. Thus, the engineered pendrin was capable of both transporting anions and generating force. C. Significance Our proposed research has high impact in hearing research. Prestin is of special interest to biologists because of its unique voltage-dependent motor function. The molecule mechanism underlying the electromotility is ascribed to the existence of a "motility actuator" conducting conformational change. The residues of the "motility actuator" and the mechanism whereby it changes the conformation of the molecule is unknown. We propose that a small peptide segment represents the eutherian EM motif. We will determine whether the domain swapping from gerbil prestin can endow the pendrin electromotility by measuring the NLC and motor functions of the "prestinlized" pendrin. Since the crystallography data of prestin or its paralogs are unavailable at present, the experimental demonstration of similarity on structural and functional features of prestin and its paralogs is very limited. Our proposed research will determine the essential motif for electromotility of prestin. That will help us to extend the knowledge of prestin's working mechanism, thus furthering our understanding of prestin structure. The electrophysiological studies of pendrin proposed in our research will also contribute the understanding of the mechanism underlying anion transport in inner ear. Many studies have addressed the prediction of amino acid sites critical for prestin motor function by investigating mutant prestin molecules. However, only change or loss of function mutations have been identified in over 100 mutants examined to date. Most of the works focused on the mechanism of prestin motility was conducted on the single amino acid level. In order to be the force generator, the mobile region should be clustered and large enough to account for the significant changes of the molecular shape (Rybalchenko and Santos-Sacchi, 2008). Our experiments are designed for the first time to examine the functional role of a sequence segment and produce a more unified view of the functional and structural relationship of prestin. In addition, we will examine the anion uptake capability to reveal the relationship of motility and transporter function. We expect a construction of prestin-pendrin chimera with both motor and transport functions. The gain of motility will provide strong support to our theoretical predictions of the EM for motility of prestin. Experimental demonstration of the transport capability retained will allow us to establish a closer connection between a structural model and the biological function of both prestin and pendrin. D. Plans Our plan for current project is to further confirm the gain of motor function of "prestinlized" pendrin. First, we will record the NLC from more cells expressing "prestinlized" pendrin. An appropriate sample size is essential for statistics. Second, substitution of EM motif from gerbil prestin conferred pendrin chimeric protein with large NLC. We will confirm whether the gain of NLC could confer "prestinlized" pendrin with motor capability. The motor function of prestin is mediated by the "actuator" in the molecule and manifested as length changes in OHCs or dimensional alternations in prestin-transfected cells. We will measure electromotility of transfected HEK cells using the microchamber technique (He et al., 1994; Zheng et al., 2000; Albert et al., 2007). Since a spherical cell cannot effectively change its surface area, we will altere the spherical shape by drawing them into the suction pipette (microchamber) to produce a dumbbell shape. An estimated voltage of 200 mV (peak-to-peak) will be applied to the extruded membrane segment. This voltage magnitude is large enough to generate a saturated response. Gerbil prestin will be used as a positive control. Prestin-transfected HEK cells exhibited large cycle-by-cycle response to the sinusoidal voltage stimulation (Tan et al., 2011). In contrast, none of 12 pendrin-transfected cells measured exhibited any somatic motility with a system resolution of 5 nm (data not shown). It is not surprising if A4(g)-tansfected HEK cell also display robust motility when the cell is stimulated with 100 Hz sinusoidal voltage burst. Pending grants. Title:Creation of Prestin paralogs with dual functions Funding Source: Deafness Research Foundation ($25,000) Principal Investigator: Jie Tang Funding Period: 7/1/2011 - 6/30/2012

项目成果

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JIE TANG其他文献

JIE TANG的其他文献

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