Characterization of a novel deafwaddler mutant implicates a role for PMCA2 regulation in the interaction with cadherin 23 and resulting auditory phenotype

Abstract

The transport of calcium ions across a cell membrane is critical for cell signaling in eukaryotic organisms. Maintenance of a low intracellular concentration is necessary for recognition of the calcium signal and, conversely, high intracellular levels of calcium are toxic. In stereocilia bundles of auditory hair cells, the plasma membrane calcium ATPase 2 (PMCA2) is the primary mechanism for calcium extrusion. Calcium carries part of the transduction current into the stereocilia and plays a role in adaptation. Furthermore, extracellular calcium in the microdomain surrounding the stereocilia is required for rigidity of cadherin 23, a component of the stereocilia tip-link encoded by the <italic>Cdh23</italic> gene. Therefore, PMCA2 is a critical player in normal function of hair cells and subsequent auditory transduction. The work described in this thesis deals primarily with understanding how variants in the <italic>Atp2b2</italic> gene, which encodes for PMCA2, and alterations in the PMCA2 pump affect auditory transduction. A novel PMCA2 null allele, <italic>dfw<super>i5</super></italic>, was characterized and used to highlight the interaction of the <italic>Atp2b2</italic> and <italic>Cdh23</italic> genes. We demonstrate that only one affected copy of <italic>Cdh23</italic> is necessary to cause broad frequency hearing impairment in +/<italic>dfw<super>i5</super></italic> mice. These mice were also used to study the role of PMCA2 function in the maturation of auditory sensitivity. We show that three weeks of age is a critical time period in the development of auditory transduction where sensitivity improves in wild-type controls but worsens at most frequencies in +/<italic>dfw<super>i5</super></italic>. Frequency dependent recovery of sensitivity occurs by 5 weeks in +/<italic>dfw<super>i5</super></italic> and correlates to preferential expression of <italic>α-Atp2b2</italic> and an up-regulation in PMCA2 in these mice. We also quantitatively compare PMCA2 function in auditory transduction using an allelic series of deafwaddler mice. Surprisingly, heterozygotes of two PMCA2 null alleles, <italic>dfw<super>2J</super></italic> and <italic>dfw<super>i5</super></italic>, have different auditory phenotypes which we attribute to differential mutant allele regulation. Higher levels of mutant <italic>Atp2b2</italic> in +/<italic>dfw<super>i5</super></italic> correlate to decreased auditory sensitivity. A key finding of this thesis is that the differential regulation of <italic>dfw<super>i5</super></italic> and <italic>dfw<super>2J</super></italic> alleles corresponds to ancestral haplotype at <italic>Atp2b2</italic>, and implicates PMCA2 regulation as a feature of age-related hearing loss in C57BL/6J.