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 InsP ₃ . 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.

Myelination is a complex process that requires coordinated Schwann cell-axon interactions during development and regeneration. Positive and negative regulators of myelination have been recently described, and can belong either to Schwann cells or neurons. Vimentin is a fibrous component present in both Schwann cell and neuron cytoskeleton, the expression of which is timely and spatially regulated during development and regeneration. We now report that vimentin negatively regulates myelination, as loss of vimentin results in peripheral nerve hypermyelination, owing to increased myelin thickness in vivo, in transgenic mice and in vitro in a myelinating co-culture system. We also show that this is due to a neuron-autonomous increase in the levels of axonal neuregulin 1 (NRG1) type III. Accordingly, genetic reduction of NRG1 type III in vimentin-null mice rescues hypermyelination. Finally, we demonstrate that vimentin acts synergistically with TACE, a negative regulator of NRG1 type III activity, as shown by hypermyelination of double Vim / Tace heterozygous mice. Our results reveal a novel role for the intermediate filament vimentin in myelination, and indicate vimentin as a regulator of NRG1 type III function.

Acidic phospholipids increase the affinity of the plasma membrane Ca(2+)-ATPase pump for Ca(2+). They interact with the C-terminal region of the pump and with a domain in the loop connecting transmembrane domains 2 and 3 (A(L) region) next to site A of alternative splicing. The contribution of the two phospholipid-binding sites and the possible interference of splicing inserts at site A with the regulation of the ATPase activity of isoform 2 of the pump by phospholipids have been analyzed. The activity of the full-length z/b variant (no insert at site A), the w/b (with insert at site A), and the w/a variant, containing both the 45-amino acid A-site insert and a C-site insert that truncates the pump in the calmodulin binding domain, has been analyzed in microsomal membranes of overexpressing CHO cells. The A-site insertion did not modify the phospholipid sensitivity of the pump, but the doubly inserted w/a variant became insensitive to acidic phospholipids, even if containing the intact A(L) phospholipid binding domain. Pump mutants in which 12 amino acids had been deleted, or single lysine mutations introduced, in the A(L) region were studied by monitoring agonist-induced Ca(2+) transients in overexpressing CHO cells. The 12-residue deletion completely abolished the ATPase activity of the w/a variant but only reduced that of the z/b variant, which was also affected by the single lysine substitutions in the same domain. A structural interpretation of the interplay of the pump with phospholipids, and of the mechanism of their activation, is proposed on the basis of molecular modeling studies.