A molecular mechanism for electrical tuning of cochlear hair cells

Science

Volumn 283, Issue 5399, 1999, Pages 215-217

  Ramanathan, Krishnan ^a   Michael, Timothy H ^a   Jiang, Guo Jian ^a   Hiel, Hakim ^a   Fuchs, Paul A ^a  

^a JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

POTASSIUM CHANNEL;

ANIMAL TISSUE; ARTICLE; AUDITORY STIMULATION; COCHLEA POTENTIAL; ELECTROSTIMULATION; GENE EXPRESSION; HAIR CELL; KINETICS; MECHANICAL STIMULATION; MOLECULAR INTERACTION; NONHUMAN; PRIORITY JOURNAL;

ALTERNATIVE SPLICING; ANIMALS; CALCIUM; CELL LINE; ELECTROPHYSIOLOGY; GENE EXPRESSION; HAIR CELLS; HUMANS; IN SITU HYBRIDIZATION; ION CHANNEL GATING; KINETICS; LARGE-CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM CHANNEL BETA SUBUNITS; LARGE-CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM CHANNELS; MEMBRANE POTENTIALS; PATCH-CLAMP TECHNIQUES; POTASSIUM CHANNELS; POTASSIUM CHANNELS, CALCIUM-ACTIVATED; QUAIL; REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION; RNA, MESSENGER; TRANSFECTION;

ANIMALIA; AVES; VERTEBRATA;

EID: 0033534539     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5399.215     Document Type: Article

References (34)

1. A. C. Crawford and R. Fettiplace, J. Physiol. 312, 377 (1981).
2. J. J. Art and R. Fettiplace, ibid. 385, 207 (1987).
3. J. Hudspeth and R. S. Lewis, ibid. 400, 237 (1988).
4. J. J. Art, Y. C. Wu, R. Fettiplace, J. Gen. Physiol. 105, 49 (1995).
5. G.-J. Jiang et al., Proc. R. Soc. London Ser. B. 264, 731 (1997).
6. P. A. Fuchs and M. G. Evans, J. Comp. Physiol. A 164, 151 (1988); P. A. Fuchs, T. Nagai, M. G. Evans, J. Neurosci. 8, 2460 (1988).
7. P. A. Fuchs and M. G. Evans, J. Comp. Physiol. A 164, 151 (1988); P. A. Fuchs, T. Nagai, M. G. Evans, J. Neurosci. 8, 2460 (1988).
8. D. S. Navaratnam, T. J. Bell, T. D. Tu, E. L. Cohen, J. C. Oberholtzer, Neuron 19, 1077 (1997).
9. K. P. Rosenblatt, Z. Sun, S. Heller, A. J. Hudspeth, ibid., p. 1061.
10. E. M. C. Jones, C. Laus, R. Fettiplace, Proc. R. Soc. London Ser. B. 265, 685 (1998).
11. T. Michael et al., Biophys. J. 72, A352 (1997).
12. J. Tseng-Crank et al., Neuron 13, 1315 (1994).
13. J. Xie and D. P. McCobb, Science 280, 443 (1998).
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. 2, and 5 mM Hepes (pH = 7.2).
16. 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. (v v1/2)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. O. B. McManus et al., Neuron 14, 645 (1995); S. I. Dworetzky et al., J. Neurosci. 16, 4543 (1996); J. Tseng-Crank et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9200 (1996); P. Meera, M. Wallner, Z. Jiang, L. Toro, FEBS Lett. 382, 84 (1996).
19. O. B. McManus et al., Neuron 14, 645 (1995); S. I. Dworetzky et al., J. Neurosci. 16, 4543 (1996); J. Tseng-Crank et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9200 (1996); P. Meera, M. Wallner, Z. Jiang, L. Toro, FEBS Lett. 382, 84 (1996).
20. O. B. McManus et al., Neuron 14, 645 (1995); S. I. Dworetzky et al., J. Neurosci. 16, 4543 (1996); J. Tseng-Crank et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9200 (1996); P. Meera, M. Wallner, Z. Jiang, L. Toro, FEBS Lett. 382, 84 (1996).
21. O. B. McManus et al., Neuron 14, 645 (1995); S. I. Dworetzky et al., J. Neurosci. 16, 4543 (1996); J. Tseng-Crank et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9200 (1996); P. Meera, M. Wallner, Z. Jiang, L. Toro, FEBS Lett. 382, 84 (1996).
22. C. Oberst et al., Oncogene 14, 1109 (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. 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. C. J. Kros, J. P. Rupersberg, A. Rüsch, Nature 394, 281 (1998).
30. P. A. Fuchs and B. H. A. Sokolowski, Proc. R. Soc. London Ser. B 241, 122 (1990).
31. M. Bers, C. W. Patton, R. Nuccitelli, Methods Cell Biol. 40, 3 (1994).
32. J. Cui, D. H. Cox, R. W. Aldrich, J. Gen. Physiol. 109, 647 (1997).
33. H. Hiel et al., Brain Res. 738, 347 (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.