NICOTINIC ACETYLCHOLINE-RECEPTOR SUBUNITS EXPRESSED IN RAT COCHLEA DETECTED BY THE POLYMERASE CHAIN-REACTION

Poly(A)(+) RNA was extracted from rat cochleae using guanidinium thioc yanate and oligo(dT)-cellulose and converted into cDNA by reverse tran scriptase using an oligo(dT) primer. Oligonucleotides complementary to conserved 5' and 3' regions of alpha and beta subunits of the neurona l nicotinic acetylcholine receptor subunit (nAChR) family were then us ed as primers to screen the cochlear cDNA via the polymerase chain rea ction (PCR) procedure. PCR products of approximately 900 bp length, pu rified by agarose gel electrophoresis, were nick translated to produce [P-32]-dCTP labelled probes for Southern Blot screening of nAChR cDNA s. Of the four alpha and three beta subunits screened, only alpha 5 an d beta 4 nAChR cDNAs hybridized. The alpha 5 PCR product was cloned an d sequenced and proved to be identical to published sequence for alpha 5. The detection of alpha 5 and beta 4 nAChR subunit expression in co chlear tissue supports previous electrophysiological and immunocytoche mical evidence for nAChR-mediated centrifugal control of hearing funct ion.