A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness.

Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The G293S and G283S mutations delayed the dissipation of Ca2+ transients induced in CHO cells by InsP3. In organotypic cultures, Ca2+ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca2+ transients induced by Ca2+ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.

Espin gene (ESPN) mutations associated with autosomal dominant hearing loss cause defects in microvillar elongation or organisation.

BACKGROUND: Espins are actin bundling proteins present in hair cell stereocilia. A recessive mutation in the espin gene (Espn) has been detected in the jerker mouse and causes deafness, vestibular dysfunction, and hair cell degeneration. More recently mutations in the human espin gene (ESPN) have been described in two families affected by autosomal recessive hearing loss and vestibular areflexia. OBJECTIVE: To report the identification of four additional ESPN mutations (S719R, D744N, R774Q, and delK848) in patients affected by autosomal dominant hearing loss without vestibular involvement. RESULTS: To determine whether the mutated ESPN alleles affected the biological activity of the corresponding espin proteins in vivo, their ability to target and elongate the parallel actin bundles of brush border microvilli was investigated in transfected LLC-PK1-CL4 epithelial cells. For three mutated alleles clear abnormalities in microvillar length or distribution were obtained. CONCLUSIONS: The results further strengthen the causative role of the espin gene in non-syndromic hearing loss and add new insights into espin structure and function.