We identified 11 human pedigrees with dominantly inherited hemolytic anemias in both the hereditary stomatocytosis and spherocytosis classes. Affected individuals in these families had an increase in membrane permeability to Na and K that is particularly marked at 0 degrees C. We found that disease in these pedigrees was associated with a series of single amino-acid substitutions in the intramembrane domain of the erythrocyte band 3 anion exchanger, AE1. Anion movements were reduced in the abnormal red cells. The 'leak' cation fluxes were inhibited by SITS, dipyridamole and NS1652, chemically diverse inhibitors of band 3. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na and K fluxes in the oocytes, and the induced Cl transport was low. These data are consistent with the suggestion that the substitutions convert the protein from an anion exchanger into an unregulated cation channel.

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Myosins have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Different members of the myosin superfamily are responsible for syndromic and nonsyndromic hearing impairment in both humans and mice. MYH14 encodes one of the heavy chains of the class II nonmuscle myosins, and it is localized within the autosomal dominant hearing impairment (DFNA4) critical region. After demonstrating that MYH14 is highly expressed in mouse cochlea, we performed a mutational screening in a large series of 300 hearing-impaired patients from Italy, Spain, and Belgium and in a German kindred linked to DFNA4. This study allowed us to identify a nonsense and two missense mutations in large pedigrees, linked to DFNA4, as well as a de novo allele in a sporadic case. Absence of these mutations in healthy individuals was tested in 200 control individuals. These findings clearly demonstrate the role of MYH14 in causing autosomal dominant hearing loss and further confirm the crucial role of the myosin superfamily in auditive functions.

Ca 2؉ 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 Ca 2؉ pulse. The G293S and G283S mutations delayed the dissipation of Ca 2؉ transients induced in CHO cells by InsP3. In organotypic cultures, Ca 2؉ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca 2؉ transients induced by Ca 2؉ 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.hereditary hearing loss ͉ mutations ͉ calcium homeostasis ͉ calcium transport T he receptive organelle of sensory hair cells in the mammalian cochlea, the stereocilia bundle, protrudes from the cell's apical surface. Inner hair cells transduce mechanical vibrations into electrical signals that are eventually transmitted to the brain to be transformed into hearing signals (1), whereas outer hair cells (OHCs) (2) amplify the vibrations of the basilar membrane (3). Mechanical stimuli that are detected as excitatory deflect a hair bundle, thus increasing tension in the tip link, a filament stretched between the tops of stereocilia. This tension is conveyed to mechanosensitive transduction (MET) channels that open to allow ions into the cell (4). The apical surface of hair cells is bathed in endolymph, which is rich in K ϩ but low in Na ϩ and Ca 2ϩ (5). K ϩ carries most the transduction current, but MET channels are Ca 2ϩ selective, i.e., Ca 2ϩ influx is significant even at the low Ca 2ϩ levels of the endolymph, which are much lower than those of other extracellular fluids (6-10): 20-23 M in the rodent cochlea (11, 12), 200-250 M in the vestibular system, possibly because of the presence there of calcium carbonate crystals (13,14). Approximately 10% of the MET current may actually be carried by Ca 2ϩ ions (15). Ca 2ϩ entering through MET channels is rapidly sequestered by buffers in the stereocilia (16,17) and is shuttled back to endolymph by the plasma membrane Ca 2ϩ pump (PMCA) (18,19), which is very concentrated in the stereocilia membrane (Ϸ2,000 per squared micrometer) (15, 19-21). The PMCA is assumed to increase Ca 2ϩ in the immediate pro...

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