All pigeon vestibular hair cells contain a mixture of different basolateral ionic currents. The proportion of each component current in the mixture determines the macroscopic ionic current profile or phenotype. In Type II hair cells, this mixture produces """"""""fast"""""""" and """"""""slow"""""""" hair cells which have different membrane filtering properties and different frequency responses. This mixture differs depending upon whether the hair cell is in the striolar or extrastriolar region of the otolithic utricular macula or whether it is on the apex or in the periphery of the semicircular canal crista. The ionic current phenotype must be important since regenerated hair cells return with the same phenotype in the same locations on the crista. This proposal also seeks to correlate the phenotype and genotype of certain major ionic current components and receptors in the same cell in different locations of the neuroepithelium. Thus, the specific aims (SAs) are to study the mRNA transcript profiles of three major currents (SA1-2) and the two types of efferent neurotransmitter receptors (SA3) that contribute to the phenotype of pigeon type II and type I vestibular hair cells. In type II hair cells, the currents that will be studied are an outward transient A-type K+ current, IKA, an inward rectifier current, IRIK1, and the receptor types, the muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors. In type I hair cells, the current that will be studied will be IKI, the dominant and ubiquitous current in all type I hair cells. Patch clamp studies of single hair cells in various regions of the epithelial slice will be combined with quantitative single cell RT-PCR studies to determine the parameters of the cells' ionic currents, its response to ACh, carbachol, mAChR and nAChR agonists and antagonists and the quantity of mRNA transcripts encoding selected ion channels and receptors of interest in the same cells. Once the cDNAs for these ion channels and receptors are cloned, each clone will be functionally verified by injecting or transfecting cRNA into two heterologous expression systems (HESs), oocytes and one of the mammalian cell lines (eg. CHO cells). Recording of ionic current in these HESs will be compared to those from the native cells. These HES studies will permit, for example, investigation of the ion channel isolated from other members of the cadre of currents found in the hair cells. It is hypothesized that the ion channel currents in HESs will resemble those of the Kv and Kir subfamilies of ion currents noted for many vertebrates. While patch clamping, single cell RT-PCR, and the slice preparation have been used separately to study single cells in other neural systems, they have not yet been used together to study the same cell in the inner ear. It is anticipated that that the use of these techniques together will allow electrophysiological and genetic studies of single hair cells in small discrete identifiable locations on the vestibular neuroepithelium.
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