Ion channels and membrane receptors on hair cells have been well- characterized pharmacologically and biophysically. However, little is known about their nature at the molecular level. Furthermore, there is evidence for the existence of at least one novel subtype of K+channel, based on electrophysiological data. In addition, individual hair cells display a variety of ionic conductances, suggesting the existence of multiple subtypes of ion channels and receptors. Molecular characterization of these receptors and ion channels at the cellular level would provide greater insight into the intracellular processes which regulate the response of hair cells, and improve our understanding of how a single neural cell can generate a unique response even though it may possess the same basic signalling equipment as its neighbor. Using a novel technique which combines electrophysiology and molecular biology, we will characterize the complement of potassium channels which are found in isolated hair cells of the auditory system, correlating these findings with the location of the cells within the cochlea and with the cells morphological features. Hair cells will be isolated from the cochlea of the guinea pig, patch-clamped and the potassium currents characterized using standard methods. Following this, the cellular contents will be aspirated into the patch pipette, which is filled with a solution containing the necessary ingredients to reverse-transcribe the cellular mRNA into cDNA. Following a series of amplification steps and radiolabeling with 32P, the RNA (amplified 10 6-fold) will be hybridized to a slot-blot membrane containing cDNAs which encode the sequences for the known mammalian K+ channels. Autoradiography will be used to determine the presence and relative abundance of the K+ channels in the individual hair cells. This technique, which combines electrophysiology and mRNA amplification, thus permits the simultaneous detection of the presence and abundance of multiple transcripts in a single cell, and should prove to be a powerful tool for future studies of the molecular biology of hair cells.
| Holt, J C; Lioudyno, M; Athas, G et al. (2001) The effect of proteolytic enzymes on the alpha9-nicotinic receptor-mediated response in isolated frog vestibular hair cells. Hear Res 152:25-42 |
