The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nervous system, the channel is expressed in neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells.
After opening in response to mechanical stimuli, hair cell transduction channels adapt with fast and slow mechanisms that each depend on Ca(2+). We demonstrate here that transduction and adaptation require phosphatidylinositol 4,5-bisphosphate (PIP(2)) for normal kinetics. PIP(2) has a striking distribution in hair cells, being excluded from the basal region of hair bundles and apical surfaces of frog saccular hair cells. Localization of a phosphatidylinositol lipid phosphatase, Ptprq, to these PIP(2)-free domains suggests that Ptprq maintains low PIP(2) levels there. Depletion of PIP(2) by inhibition of phosphatidylinositol 4-kinase or sequestration by aminoglycosides reduces the rates of fast and slow adaptation. PIP(2) and other anionic phospholipids bind directly to the IQ domains of myosin-1c, the motor that mediates slow adaptation, permitting a strong interaction with membranes and likely regulating the motor's activity. PIP(2) depletion also causes a loss in transduction current. PIP(2) therefore plays an essential role in hair cell adaptation and transduction.
Ca2+ signaling serves distinct purposes in different parts of a hair cell. The Ca 2+ concentration in stereocilia regulates adaptation and, through rapid transduction-channel reclosure, underlies ampli®ca-tion of mechanical signals. In presynaptic active zones, Ca 2+ mediates the exocytotic release of afferent neurotransmitter. At efferent synapses, Ca 2+ activates the K + channels that dominate the inhibitory postsynaptic potential. A copious supply of diffusible protein buffer isolates the three signals by restricting the spread of free Ca 2+ and limiting the duration of its action. Using cDNA subtraction and a gene expression assay based on in situ hybridization, we detected abundant expression of mRNAs encoding the Ca 2+ buffer parvalbumin 3 in bullfrog saccular and chicken cochlear hair cells. We cloned cDNAs encoding this protein from the corresponding inner-ear libraries and raised antisera against recombinant bullfrog parvalbumin 3. Immunohistochemical labeling indicated that parvalbumin 3 is a prominent Ca 2+ -binding protein in the compact, cylindrical hair cells of the bullfrog's sacculus, and occurs as well in the narrow, peanut-shaped hair cells of that organ. Using quantitative Western blot analysis, we ascertained that the concentration of parvalbumin 3 in saccular hair cells is approximately 3 mM. Parvalbumin 3 is therefore a signi®cant mobile Ca 2+ buffer, and perhaps the dominant buffer, in many types of hair cell. Moreover, parvalbumin 3 provides an early marker for developing hair cells in the frog, chicken, and zebra®sh.
PRDM16 (positive regulatory domain 16) is localized in the critical region for cardiomyopathy in patients with deletions of chromosome 1p36, as defined by Gajecka et al., American Journal of Medical Genetics, 2010, 152A, 3074–3083, and encodes a zinc finger transcription factor. We present the first fetal case of left ventricular non‐compaction (LVNC) with a PRDM16 variant. The third‐trimester obstetric ultrasound revealed a hydropic fetus with hydramnios and expanded hypokinetic heart. After termination of pregnancy, foetopathology showed a eutrophic fetus with isolated cardiomegaly. Endocardial fibroelastosis was associated with non‐compaction of the myocardium of the left ventricle. Exome sequencing (ES) identified a de novo unreported p.(Gln353*) heterozygous nonsense variant in PRDM16. ES also identified two rare variants of unknown significance, according to the American College of Medical Genetics and Genomics guidelines, in the titin gene (TTN): a de novo missense p.(Lys14773Asn) variant and a c.33043+5A>G variant inherited from the mother. Along with the PRDM16 de novo probably pathogenic variant, TTN VOUS variants could possibly contribute to the severity and early onset of the cardiac phenotype. Because of the genetic heterogeneity of cardiomyopathies, large panels or even ES could be considered as the main approaches for the molecular diagnosis, particularly in fetal presentations, where multiple hits seem to be common.
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