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An In Vitro Study on Prestin Analog Gene in the Bullfrog Hearing Organs.
Wang Z
,
Qian M
,
Wang Q
,
Liu H
,
Wu H
,
Huang Z
.
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The prestin-based active process in the mammalian outer hair cells (OHCs) is believed to play a crucial role in auditory signal amplification in the cochlea. Prestin belongs to an anion transporter family (SLC26A). It is densely expressed in the OHC lateral plasma membrane and functions as a voltage-dependent motor protein. Analog genes can be found in the genome of nonmammalian species, but their functions in hearing are poorly understood. In the present study, we used the gerbil prestin sequence as a template and identified an analog gene in the bullfrog genome. We expressed the gene in a stable cell line (HEK293T) and performed patch-clamp recording. We found that these cells exhibited prominent nonlinear capacitance (NLC), a widely accepted assay for prestin functioning as a motor protein. Upon close examination, the key parameters of this NLC are comparable to that conferred by the gerbil prestin, and nontransfected cells failed to display NLC. Lastly, we performed patch-clamp recording in HCs of all three hearing organs in bullfrog. HCs in both the sacculus and the amphibian papilla exhibited a capacitance profile that is similar to NLC while HCs in the basilar papilla showed no sign of NLC. Whether or not this NLC-like capacitance change is involved in auditory signal amplification certainly requires further examination; our results represent the first and necessary step in revealing possible roles of prestin in the active hearing processes found in many nonmammalian species.
Figure 1. Alignment of amino acid sequences of SLC26A5 of mouse, gerbil, Xenopus, and bullfrog. Different colors had been used to represent identity of each residue among four species. Red block: full identity at a residue; red letter: partial identity at a residue; black: complete disparity at a residue. Gaps in the aligned sequences were indicated by the dashed line.
Figure 2. Nonlinear capacitance obtained from gPres- and fPres-transfected cells and a mouse OHC. (a) Confocal microscopy images of HEK cells transfected by gPres and fPres. (b) OHC patch. (c) Whole-cell currents of gPres- and fPres-transfected cells and OHC. Cells were held at -80 mV for current recordings. Voltage steps (300 ms in duration) varied from -150 to 100 mV in 10 mV steps. Black-gPres, red-fPres, blue-OHC. (d) NLC obtained from gPres- and fPres-transfected cells. Black-gPres, red-fPres. NLC obtained from the mouse OHC, blue curve. (e) This one showed the lack of detectable NLC in a representative control cell.
Figure 3. NLC functions of fPres, gPres, and mouse OHC. (a–d) Showed four parameters derived from curve fittings with Boltzmann's function for fPres (n = 16), gPres (n = 15), and OHC (n = 6). Data were expressed as mean ± s.d. ∗P < 0.05, ∗∗P < 0.01.
Figure 4. Images of frog's hearing organ. (a) Dissection of the frog's inner ear which contained three auditory organs (AP, BP, and S) under a 10x microscope. (b, c) Displayed was a higher magnification image of the AP under a 100x and 600x microscope. (d, e) Displayed was a higher magnification image of the BP under a 100x and 600x microscope. (f, g) Displayed was a higher magnification image of the S under a 100x and 600x microscope.
Figure 5. NLC functions of frog's three auditory organs (AP, BP, and S). (a) NLC obtained from the hair cells of three auditory organs. Red: the amphibian papilla (AP); black: the sacculus (S); blue: the basilar papilla (BP). No NLC was detected in the hair cells of the basilar papilla (BP). (b) Four parameters derived from curve fittings with Boltzmann's function for AP (n = 10) and S (n = 8). Mouse OHC was used as a comtrol. Data are expressed as mean ± s.d. ∗∗P < 0.01, ∗P < 0.05 (Student's t-test).
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