The Novel Mouse Mutation Oblivion Inactivates the PMCA2 Pump and Causes Progressive Hearing Loss

Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU (N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion (allele symbol Obl), showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C→T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca2+ pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca2+ exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone

Repressor element 1-silencing transcription factor deficiency yields profound hearing loss through Kv7.4 channel upsurge in auditory neurons and hair cells.

Repressor element 1-silencing transcription factor (REST) is a transcriptional repressor that recognizes neuron-restrictive silencer elements in the mammalian genomes in a tissue- and cell-specific manner. The identity of REST target genes and molecular details of how REST regulates them are emerging. We performed conditional null deletion of Rest (cKO), mainly restricted to murine hair cells (HCs) and auditory neurons (aka spiral ganglion neurons (SGNs)). Null-inactivation of full-length REST did not affect the development of normal HCs and SGNs but manifested as progressive hearing loss in adult mice. We found that the inactivation of REST resulted in an increased abundance of Kv7.4 channels at the transcript, protein, and functional levels. Specifically, we found that SGNs and HCs from Rest cKO mice displayed increased Kv7.4 expression and augmented Kv7 currents; SGN’s excitability was also significantly reduced. Administration of a compound with Kv7.4 channel activator activity, fasudil, recapitulated progressive hearing loss in mice. In contrast, inhibition of the Kv7 channels by XE991 rescued the auditory phenotype of Rest cKO mice. Previous studies identified some loss-of-function mutations within the Kv7.4-coding gene, Kcnq4, as a causative factor for progressive hearing loss in mice and humans. Thus, the findings reveal that a critical homeostatic Kv7.4 channel level is required for proper auditory functions

Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations

Mutations in Myo7a cause hereditary deafness in mice and humans. We describe the effects of two mutations, Myo7a6J and Myo7a4626SB, on mechano-electrical transduction in cochlear hair cells. Both mutations result in two major functional abnormalities that would interfere with sound transduction. The hair bundles need to be displaced beyond their physiological operating range for mechanotransducer channels to open. Transducer currents also adapt more strongly than normal to excitatory stimuli. We conclude that myosin VIIA participates in anchoring and holding membrane-bound elements to the actin core of the stereocilium. Myosin VIIA is therefore required for the normal gating of transducer channels

PMCA2w/a Splice Variant: A Key Regulator of Hair Cell Mechano-transduction Machinery

Sensory hair cells of the inner ear detect sound stimuli, inertial or gravi- tational forces. These mechanical inputs cause de\ufb02ection of the cell stereociliary bundle and activate a small number of cation-selective mechano-transduction (MET) channels that admit K+ and Ca2+ ions into the cytoplasm. Stereociliary Ca2+ levels are homeostatically regulated by an unusual splicing isoform (w/a) of plasma membrane calcium-pump isoform 2 (PMCA2w/a), ablation or missense mutations of which cause deafness and loss of balance in humans and mice. At variance with other PMCA2 isoforms, PMCA2w/a expressed in CHO transfectants increases only marginally its activity in response to a rapid increase of the cytoplasmic free Ca2+ concentration ([Ca2+]c). In this expression system, deafness-related mutations of PMCA2w/a decrease the pump ability to extrude Ca2+ both at steady state and in response to a [Ca2+]c rise. Consistent with these \ufb01ndings, mouse strains in which the pump is genetically ablated or mutated show hearing impairment correlated with defects in homeostatic regulation of stereociliary Ca2+, decreased sensitivity of the MET channels to hair bundle displacement, and morphological abnormalities in the organ of Corti. These results highlight a critical role played by PMCA2w/a in the control of hair cell function and survival and provide mechanistic insight into the etiology of deafness and vestibular disorders