112 research outputs found

    Volume-regulated Cl- current: contributions of distinct Cl- channel and localized Ca2+ signals.

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    The swelling-activated chloride current (ICl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of ICl,swell is the volume regulated anion channel (VRAC). LRRC8A (SWELL1) was recently identified as an essential component of VRAC but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl- channels, such as anoctamin 1 (ANO1) were also suggested to contribute to ICl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of ICl,swell; we also explored the role of intracellular Ca2+ in ICl,swell activation. We used CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to ICl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca2+ transients and these Ca2+ signals were required to activate both, the LRRC8A- and ANO1-dependent components of ICl,swell. Both ICl,swell components required localized rather than global Ca2+ for activation. Interestingly, while intracellular Ca2+ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca2+ transients linked to the ICl,swell activation were mediated by the GPCR-independent PLC isoforms

    Molecular Etiology of Hearing Impairment in Inner Mongolia: mutations in SLC26A4 gene and relevant phenotype analysis

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    <p>Abstract</p> <p>Background</p> <p>The molecular etiology of hearing impairment in Chinese has not been thoroughly investigated. Study of <it>GJB2 </it>gene revealed that 30.4% of the patients with hearing loss in Inner Mongolia carried <it>GJB2 </it>mutations. The <it>SLC26A4 </it>gene mutations and relevant phenotype are analyzed in this study.</p> <p>Methods</p> <p>One hundred and thirty-five deaf patients were included. The coding exons of <it>SLC26A4 </it>gene were sequence analyzed in 111 patients, not including 22 patients carrying bi-allelic <it>GJB2 </it>mutations or one patient carrying a known <it>GJB2 </it>dominant mutation as well as one patient with <it>mtDNA </it>1555A>G mutation. All patients with <it>SLC26A4 </it>mutations or variants were subjected to high resolution temporal bone CT scan and those with confirmed enlarged vestibular aqueduct and/or other inner ear malformation were then given further ultrasound scan of thyroid and thyroid hormone assays.</p> <p>Results</p> <p>Twenty-six patients (19.26%, 26/135) were found carrying <it>SLC26A4 </it>mutation. Among them, 17 patients with bi-allelic <it>SLC26A4 </it>mutations were all confirmed to have EVA or other inner ear malformation by CT scan. Nine patients were heterozygous for one <it>SLC26A4 </it>mutation, including 3 confirmed to be EVA or EVA and Mondini dysplasia by CT scan. The most common mutation, IVS7-2A>G, accounted for 58.14% (25/43) of all <it>SLC26A4 </it>mutant alleles. The shape and function of thyroid were confirmed to be normal by thyroid ultrasound scan and thyroid hormone assays in 19 of the 20 patients with EVA or other inner ear malformation except one who had cystoid change in the right side of thyroid. No Pendred syndrome was diagnosed.</p> <p>Conclusion</p> <p>In Inner Mongolia, China, mutations in <it>SLC26A4 </it>gene account for about 12.6% (17/135) of the patients with hearing loss. Together with <it>GJB2 </it>(23/135), <it>SLC26A4 </it>are the two most commonly mutated genes causing deafness in this region. Pendred syndrome is not detected in this deaf population. We established a new strategy that detects <it>SLC26A4 </it>mutations prior to the temporal bone CT scan to find EVA and inner ear malformation patients. This model has a unique advantage in epidemiologic study of large deaf population.</p

    Inflammatory mediator bradykinin increases population of sensory neurons expressing functional T-type Ca2+ channels

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    T-type Ca2+ channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca2+ currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca2+ channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca2+ currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect CaV3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a 'reserve pool' of CaV3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions