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Keywords:

  • in situ hybridization;
  • organ of Corti;
  • outer hair cell cDNA library;
  • potassium channels;
  • RT-PCR

Abstract

Physiological evidence suggests that SK-type Ca2+-activated K+ channels participate in ACh-induced hyperpolarization of OHCs (outer hair cells). Based on the sequences published by Köhler et al. (1996, Science, 273: 1709), we designed degenerated primers recognizing cDNA subunits of rSK1, rSK2 and rSK3. Using this concensus set of primers, we probed by PCR a rat organ of Corti cDNA library. Two PCR products of 707 base pairs with sequence identical to rSK3 and rSK2 were obtained and cloned to generate RNA probes for in situ hybridization in the rat cochlea. The subunit rSK2 showed hybridization in the organ of Corti, at the location of the OHCs. The expression of rSK2 by OHCs was confirmed by probing with PCR a poly(A) amplified OHC cDNA library. During development, rSK2 hybridization in the organ of Corti was negative at embryonic days E16, E18 and at P0, weak at P4 and stronger from P8 to adulthood. The subunit rSK2 could also be detected in the spiral ganglion from P4 to the adult stage. Contrary to rSK2, the subunit rSK3 did not show specific hybridization in the organ of Corti at the adult stage (P120) and only a weak expression was observed at P10 and P21. Our study demonstrates expression of rSK2 in OHCs. These potassium channels are good candidates to underlie the ACh-activated K+ currents recorded during patch-clamp recordings in isolated OHCs. The expression of rSK2 in the cochlear ganglion at the adult stage suggests that SK Ca2+-activated K+ channels may also participate in the repolarization of the auditory neurons after the action potential and may influence their firing patterns.