Nicotinic acetylcholine receptor subunits expressed in rat cochlea detected by the polymerase chain reaction

Abstract

Poly(A) + RNA was extracted from rat cochleae using guanidinium thiocyanate and oligo(dT)-cellulose, and converted into cDNA by reverse transcriptase using an oligo(dT) primer. Oligonucleotides complementary to conserved 5′ and 3′ regions of α and β subunits of the neuronal nicotinic acetylcholine receptor subunit (nAChR) family were then used as primers to screen the cochlear cDNA via the polymerase chain reaction (PCR) procedure. PCR products of approximately 900 bp length, purified by agarose gel electrophoresis, were nick translated to produce [ 32P]-dCTP labelled probes for Southern Blot screening of nAChR cDNAs. Of the four α and three β subunits screened, only α5 and β4 nAChR cDNAs hybridized. The α5 PCR product was cloned and sequenced and proved to be identical to published sequence for α5. The detection of α5 and β4 nAChR subunit expression in cochlear tissue supports previous electrophysiological and immunocytochemical evidence for nAChR-mediated centrifugal control of hearing function.