A Molecular Mechanism for Electrical Tuning of Cochlear Hair Cells
10.1126/science.283.5399.215
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Cochlear frequency selectivity in lower vertebrates arises in part from electrical tuning intrinsic to the sensory hair cells. The resonant frequency is determined largely by the gating kinetics of calcium-activated potassium (BK) channels encoded by the slo gene. Alternative splicing of slo from chick cochlea generated kinetically distinct BK channels. Combination with accessory β subunits slowed the gating kinetics of α splice variants but preserved relative differences between them. In situ hybridization showed that the β subunit is preferentially expressed by low-frequency (apical) hair cells in the avian cochlea. Interaction of β with α splice variants could provide the kinetic range needed for electrical tuning of cochlear hair cells.

Bibliography

Ramanathan, K., Michael, T. H., Jiang, G.-J., Hiel, H., & Fuchs, P. A. (1999). A Molecular Mechanism for Electrical Tuning of Cochlear Hair Cells. Science, 283(5399), 215–217.

Authors 5
  1. Krishnan Ramanathan (first)
  2. Timothy H. Michael (additional)
  3. Guo-Jian Jiang (additional)
  4. Hakim Hiel (additional)
  5. Paul A. Fuchs (additional)
References 34 Referenced 163
  1. Crawford A. C., Fettiplace R., J. Physiol. 312, 377 (1981). (10.1113/jphysiol.1981.sp013634) / J. Physiol. by Crawford A. C. (1981)
  2. Art J. J., Fettiplace R., ibid. 385, 207 (1987). / ibid. by Art J. J. (1987)
  3. J. Hudspeth and R. S. Lewis ibid. 400 237 (1988). (10.1113/jphysiol.1988.sp017119)
  4. Art J. J., Wu Y. C., Fettiplace R., J. Gen. Physiol. 105, 49 (1995). (10.1085/jgp.105.1.49) / J. Gen. Physiol. by Art J. J. (1995)
  5. Jiang G.-J., et al., Proc. R. Soc. London Ser. B. 264, 731 (1997). (10.1098/rspb.1997.0104) / Proc. R. Soc. London Ser. B. by Jiang G.-J. (1997)
  6. Fuchs P. A., Evans M. G., J. Comp. Physiol. A 164, 151 (1988); (10.1007/BF00603947) / J. Comp. Physiol. A by Fuchs P. A. (1988)
  7. Fuchs P. A., Nagai T., Evans M. G., J. Neurosci. 8, 2460 (1988). (10.1523/JNEUROSCI.08-07-02460.1988) / J. Neurosci. by Fuchs P. A. (1988)
  8. Navaratnam D. S., Bell T. J., Tu T. D., Cohen E. L., Oberholtzer J. C., Neuron 19, 1077 (1997). (10.1016/S0896-6273(00)80398-0) / Neuron by Navaratnam D. S. (1997)
  9. K. P. Rosenblatt Z. Sun S. Heller A. J. Hudspeth ibid. p. 1061.
  10. Jones E. M. C., Laus C., Fettiplace R., Proc. R. Soc. London Ser. B. 265, 685 (1998). (10.1098/rspb.1998.0348) / Proc. R. Soc. London Ser. B. by Jones E. M. C. (1998)
  11. Michael T., et al., Biophys. J. 72, A352 (1997). / Biophys. J. by Michael T. (1997)
  12. Tseng-Crank J., et al., Neuron 13, 1315 (1994). (10.1016/0896-6273(94)90418-9) / Neuron by Tseng-Crank J. (1994)
  13. 10.1126/science.280.5362.443
  14. cSlo1 was cloned from a chick cochlear cDNA library (5). PCR of the cochlear cDNA library and RT-PCR of cochlear tissue identified the 61–amino acid insert. A chimeric construct incorporating the 61–amino acid insert was made by restriction digest and ligation of the PCR product with cSlo1 and was packaged into pcDNA3.1 vector (Invitrogen San Diego CA) for transfection into mammalian cells.
  15. HEK293 cells were transiently transfected by calcium phosphate precipitation. Inside-out patches (IOPs) were excised and analyzed with standard voltage clamp protocols (5). Ionic conditions were symmetrical with the exception of buffered calcium and contained 140 mM KCl 0.5 mM MgCl 2 and 5 mM Hepes (pH = 7.2).
  16. “Cytoplasmic” calcium was changed by perfusing IOPs with solutions buffered with 2 mM of EGTA Br 2 BAPTA or nitrilotriacetic acid [the free calcium concentration was determined with MaxChelator software (27)]. These concentrations were measured with a calcium electrode (Microelectrodes Bedford NH) calibrated with standard solutions obtained from World Precision Instruments (Sarasota FL).
  17. Half-activation voltages were determined by fitting normalized conductance ( g / g max ) (Figs. 1C and 2 B and D) with a Boltzmann function given by (% g/g max = 100/[1 + e −( V − V 1/2 ) qF / RT ]) where q is the gating charge F is Faraday's constant R is the universal gas constant and T is the absolute temperature.
  18. McManus O. B., et al., Neuron 14, 645 (1995); (10.1016/0896-6273(95)90321-6) / Neuron by McManus O. B. (1995)
  19. Dworetzky S. I., et al., J. Neurosci. 16, 4543 (1996); (10.1523/JNEUROSCI.16-15-04543.1996) / J. Neurosci. by Dworetzky S. I. (1996)
  20. Tseng-Crank J., et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9200 (1996); (10.1073/pnas.93.17.9200) / Proc. Natl. Acad. Sci. U.S.A. by Tseng-Crank J. (1996)
  21. Meera P., Wallner M., Jiang Z., Toro L., FEBS Lett. 382, 84 (1996). (10.1016/0014-5793(96)00151-2) / FEBS Lett. by Meera P. (1996)
  22. Oberst C., et al., Oncogene 14, 1109 (1997). (10.1038/sj.onc.1200930) / Oncogene by Oberst C. (1997)
  23. Ratios of α cDNA to β cDNA were varied from 1:1 to 1:10 with no differences seen implying that the effect of β was saturated at the lowest ratio. In most experiments a ratio of 1:2 was used.
  24. In these experiments activation rates were slightly faster upon β combination. This observation is consistent with the hypothesis that β subunits principally affect channel open states with lesser influence on closed-to-open transitions. Activation steps were made from negative voltages at which the channels were fully closed. Symmetrical slowing of activation and deactivation by β would be expected for small excursions about a voltage level at which some fraction of channels are in the open state as observed in hair cells (4).
  25. K D and δ were determined from the dependence of V 1/2 on [Ca 2+ ] (Fig. 1D) where V 1/2 = −(2.303 RT /2δ F )log[Ca 2+ ] + (2.303 RT /2δ F )log[ K D ] (28). δ is the electrical distance traveled by calcium into the membrane; it varied between 0.3 and 0.5.
  26. RT-PCR was performed on quail cochlear RNA with the use of primers flanking the untranslated regions of slo -β. A full-length product (800 bp) was subcloned and sequenced to confirm its identity.
  27. Frozen tissue sections of fixed and decalcified quail temporal bone (16 μm thick) were hybridized with digoxigenin-labeled cRNA (29) obtained by in vitro transcription of the full-length RT-PCR product of slo -β from quail cochlea. Alkaline phosphatase (AP)–conjugated sheep antibody to digoxigenin was used to detect cRNA-mRNA hybrids. The labeling was visualized with the AP substrates 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate. All reagents were purchased from Boehringer-Mannheim (Indianapolis IN).
  28. The gradient in β expression as well as the fact that BK channels are fewest in apical hair cells (2 6) implies that the ratio of β to α is highest in apical hair cells and falls toward the high-frequency base. The stoichiometry of αβ combination was not explicitly tested in these experiments (19).
  29. Kros C. J., Rupersberg J. P., Rüsch A., Nature 394, 281 (1998). (10.1038/28401) / Nature by Kros C. J. (1998)
  30. Fuchs P. A., Sokolowski B. H. A., Proc. R. Soc. London Ser. B 241, 122 (1990). (10.1098/rspb.1990.0075) / Proc. R. Soc. London Ser. B by Fuchs P. A. (1990)
  31. Bers M., Patton C. W., Nuccitelli R., Methods Cell Biol. 40, 3 (1994). (10.1016/S0091-679X(08)61108-5) / Methods Cell Biol. by Bers M. (1994)
  32. Cui J., Cox D. H., Aldrich R. W., J. Gen. Physiol. 109, 647 (1997). (10.1085/jgp.109.5.647) / J. Gen. Physiol. by Cui J. (1997)
  33. Hiel H., et al., Brain Res. 738, 347 (1996). (10.1016/S0006-8993(96)01046-3) / Brain Res. by Hiel H. (1996)
  34. We thank C. Oberst and K. Bister for their gift of quail slo -β. This work was supported by grant DC00276 from the National Institute of Deafness and Communication Disorders.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:50 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 12:08 a.m.)
Indexed 2 months, 2 weeks ago (June 15, 2025, 1:21 p.m.)
Issued 26 years, 7 months ago (Jan. 8, 1999)
Published 26 years, 7 months ago (Jan. 8, 1999)
Published Print 26 years, 7 months ago (Jan. 8, 1999)
Funders 0

None

@article{Ramanathan_1999, title={A Molecular Mechanism for Electrical Tuning of Cochlear Hair Cells}, volume={283}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.283.5399.215}, DOI={10.1126/science.283.5399.215}, number={5399}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Ramanathan, Krishnan and Michael, Timothy H. and Jiang, Guo-Jian and Hiel, Hakim and Fuchs, Paul A.}, year={1999}, month=jan, pages={215–217} }