MOLECULAR DELINEATION OF THE COCHLEAR HAIR CELLS

  • Beisel, Kirk, FATHER FLANAGAN'S BOYS' HOME (PI)

Project: Research project

Project Details

Description

Our long term goal is to identify the voltage-gated ion channels expressed
in the cochlear hair cells. The specific aims of this project are to
identify and localize the expression of the voltage-gated potassium,
calcium and sodium ion channels in rat inner hair cells (IHCs) and outer
hair cells (OHCs). Once identified, these specific ion channels will be
examined in a Xenopus oocyte expression system for their
electrophysiologic and pharmacologic properties.

The first specific goal of this project is to examine unidirectional
lambdaZAP cDNA libraries, constructed from rat cochlear polyA+ RNA and
from amplified polyA+ RNA from rat IHCc and OHCs, for the presence of
cDNAs that represent transcripts of the voltage-gated ion channel proteins
present in hair cells. Gene family-specific polymerase chain reaction
(gfs-PCR) and gene specific-PCR will be used to characterize the isoform
and/or subunit status of the potassium, calcium and sodium voltage-gated
ion channels. Conserved gene family-specific primer motifs will be used
to amplify cDNAs coding for proteins predicted to be present in the
cochlear hair cells. Isoforms will be identified using a combination of
PCR product size, restriction enzyme site differences (i.e., RFLP-PCR),
hybridization studies, and dideoxy nucleotide sequence analyses. New or
novel ion channel cDNA will be characterized by obtaining a complete full
length sequence. The expressed isoforms will be analyzed by examining the
PCR products containing the 3'NT and/or 5' ends of these messages for
their DNA sequences that will be used to confirm their unique isoform
status. Gene specific probes will be developed and used in Northern-blot
analysis and in situ hybridization studies to distinguish the expression,
tissue distribution, cellular localization of the IHC and/or OHC isoforms
from other known isoforms. Once the isoforms and/or subunits expressed in
the IHCs and OHCs are established, the next goal will be to confirm the
existence of transcripts in the cochlear hair cells using in situ
hybridization. Their precise cellular location and intracellular
distribution will be determined using immunocytochemistry. The final goal
will be to examine this ion channel for their electrophysiologic and
pharmacologic properties in Xenopus oocytes.

Collectively the molecular biology and expression studies will be used to
define and establish the role of these specific ion channels in cochlear
homeostasis and in the electrophysiology of IHCs and OHCs for acoustic
signal transduction in the cochlea. These data will increase our
understanding of the molecular processes underlying hearing and its
disorders, especially regards to the functioning of cochlear hair cells as
acoustic receptors.
StatusFinished
Effective start/end date1/1/9512/31/97

Funding

  • National Institute on Deafness and Other Communication Disorders
  • National Institute on Deafness and Other Communication Disorders
  • National Institute on Deafness and Other Communication Disorders

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