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* Applied Physics Graduate Program, Department of Bioengineering, Rice University, Houston, Texas; Bobby R. Alford Department of Otolaryngology – Head and Neck Surgery, Huffington Center on Aging, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas; and ¶ Department of Bioengineering, University of California, Riverside, California
Correspondence: Address reprint requests and inquiries to Bahman Anvari, Tel.: 951-827-5726; E-mail: anvari{at}engr.ucr.edu.
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ABSTRACT |
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Cochlear outer hair cells (OHCs) exhibit electrically induced movements known as electromotility, which allows for the sensitivity and frequency-resolving capability of mammalian hearing (1
–3). The interaction between the OHC lateral wall plasma membrane and the transmembrane protein, prestin, is thought to play a central role in electromotility (4). Prestin is essential for a nonlinear capacitance (NLC), which is widely accepted as the electrical signature of electromotility (5). However, the relationship between NLC and electromotility, and the mechanistic role of prestin in either of these processes, is unknown. Since electromotility involves the coupling of electrical and mechanical properties of the membrane, the role of prestin in plasma membrane mechanics and electromechanical force (EMF) generation is vital to the understanding of this process.
Membrane tethers provide a convenient method to study membrane mechanics. Using a combined optical tweezers and whole-cell voltage-clamping system to control membrane potential (6), we have investigated the contribution of prestin to membrane mechanics and EMF generation using membrane tethers formed from three test groups consisting of: 1), untransfected, 2), wild-type (WT) prestin-transfected, and 3), single point mutant (A100W) prestin-transfected human embryonic kidney (HEK) cells (see Supplementary Material). Our choice of mutant was based on an earlier study in which replacement of an alanine residue by tryptophan (A100W), in the region of the conserved sulfate anion transporter motif resulted in a complete loss of NLC (7). Comparisons of force measurements with membrane capacitance measurements would allow us to investigate the relationships among NLC, membrane mechanics, and EMF generation.
Procedures for mechanics and EMF measurements were essentially as previously described (6,8). (See Supplementary Material for details of methods.) First, a tether was pulled at a constant rate until the force would approach a peak force Fpk. The movement was then halted and the tether was maintained at nearly constant length for several minutes, allowing the tether force to relax to a non-zero equilibrium force Feq. Effective tether viscosity (
eff), which represents the overall viscous dissipation during tether formation (8,9), and steady-state force extrapolated to zero pulling rate Fss(0), which is the force required to hold a tether at static equilibrium (9), were estimated from the measurements of tether force at different pulling rates. For EMF measurements, once Feq was attained, the patch-clamp was used to change the transmembrane potential by applying a 1-Hz sinusoid voltage wave. Forces were measured by optical monitoring of the trapped bead displacement (6). Procedures for membrane capacitance measurements are described in Supplementary Material.
Typical NLC curves for different test groups are shown in Fig. 1 (see Supplementary Material for method). WT prestin-transfected cells show a voltage-dependent NLC function similar to that observed on OHCs (4), whereas control untransfected HEK cells have a nearly constant capacitance. Consistent with an earlier study (7), a mutation (A100W) in the sulfate transporter motif region of prestin eliminates prestin-associated charge transfer by exhibiting a non-voltage-dependent capacitance function similar to that for untransfected HEK cells.
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eff between different test groups (Fig. 2 B). It is widely considered that eff is dominated by interactions between the membrane and the cytoskeleton (9). Our results therefore suggest that membrane-cytoskeleton interactions are not affected by the presence or absence of either WT prestin or prestin A100W in the membrane. WT prestin enhances EMF twofold compared to control HEK cells (Fig. 3). On the other hand, cells transfected with prestin mutant A100W show comparable EMF to untransfected controls. This finding directly correlates with our earlier observations of linear capacitance in cells transfected with this mutant. Our results therefore directly link the presence of a specific motif within prestin (and not merely the presence of prestin) to membrane electromotility.
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Salicylate, the anionic amphipathic metabolite of aspirin, has been shown to block electromotility and charge movement in prestin-transfected HEK cells (4) but does not have an effect on membrane mechanics (10,11). These results (not repeated by our study) are consistent with our model that prestin-associated EMF enhancement is closely related to charge transfer but has little relationship with membrane mechanics. Further, salicylate also blocks electromotility of normal HEK cells by changing membrane surface charge (10,12).
While the presence of prestin appears to change membrane mechanical properties such as curvature, bending stiffness, and tension, and greatly enhances charge movement in and out of the membrane, a mutation in the region of sulfate transporter motif eliminates NLC and reduces EMF without affecting membrane mechanical properties. Based on these results, we propose synergistic effects of prestin and the membrane in the generation of NLC and electromotility.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from the National Institute of Health (No. R01-DC-2775, No. R90 DK071504), the National Science Foundation (No. BES-0522862), and the Keck Center for Interdisciplinary Bioscience Training.
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FOOTNOTES |
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Submitted on February 23, 2007; accepted for publication April 20, 2007.
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REFERENCES |
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2. Brownell, W. E., A. A. Spector, R. M. Raphael, and A. S. Popel. 2001. Micro- and nanomechanics of the cochlear outer hair cell. Annu. Rev. Biomed. Eng. 3:169–194.[CrossRef][Medline]
3. Brownell, W. E. 2006. The piezoelectric outer hair cell. In Vertebrate Hair Cells. R. A. Eatock, editor. In The Springer Handbook of Auditory Research. A. N. Popper and R. R. Fay, editors. Springer, New York. 313–347.
4. Zheng, J., W. Shen, D. Z. He, K. B. Long, L. D. Madison, and P. Dallos. 2000. Prestin is the motor protein of cochlear outer hair cells. Nature. 405:149–155.[CrossRef][Medline]
5. Santos-Sacchi, J. 1991. Reversible inhibition of voltage-dependent outer hair cell motility and capacitance. J. Neurosci. 11:3096–3110.[Abstract]
6. Qian, F., S. Ermilov, D. R. Murdock, W. E. Brownell, and B. Anvari. 2004. Combining optical tweezers and patch-clamp for studies of cell membrane electromechanics. Rev. Sci. Instr. 75:2937–2942.[CrossRef]
7. Rajagopalan, L., N. Patel, S. Madabushi, J. A. Goddard, V. Anjan, F. Lin, C. Shope, B. Farrell, O. Lichtarge, A. L. Davidson, W. E. Brownell, and F. A. Pereira. 2006. Essential helix interactions in the anion transporter domain of prestin revealed by evolutionary trace analysis. J. Neurosci. 26:12727–12734.
8. Li, Z., B. Anvari, M. Takashima, P. Brecht, J. H. Torres, and A. N. D. W. E. Brownell. 2002. Membrane tether formation from outer hair cells with optical tweezers. Biophys. J. 82:1386–1395.
9. Hochmuth, R. M., J. Y. Shao, J. Dai, and M. P. Sheetz. 1996. Deformation and flow of membrane into tethers extracted from neuronal growth cones. Biophys. J. 70:358–369.
10. Zhang, P. C., A. M. Keleshian, and F. Sachs. 2001. Voltage-induced membrane movement. Nature. 413:428–432.[CrossRef][Medline]
11. Ermilov, S., D. R. Murdock, D. E. Daye, W. E. Brownell, and B. Anvari. 2005. Effects of salicylate on plasma membrane mechanics. J. Neurophysiol. 94:2105–2110.
12. McLaughlin, S. 1973. Salicylates and phospholipid bilayer membranes. Nature. 203:234.
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  M. Drexl, M. M. Mellado Lagarde, J. Zuo, A. N. Lukashkin, and I. J. Russell The Role of Prestin in the Generation of Electrically Evoked Otoacoustic Emissions in Mice J Neurophysiol, April 1, 2008; 99(4): 1607 - 1615. [Abstract] [Full Text] [PDF]
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) and untransfected HEK cells (
) are observed.
