Screening And Mechanism Study Of Fluoride-sensitive ClC Chloride Channels

Dental fluorosis is special developmental disabilities as a result of excess fluorideingestion during tooth formation and mineralization, mainly involving the enamel ofpermanent teeth. A white horizontal lines running across the surfaces of the teeth, whiteopaque appearance of the enamel, yellow to light brown staining of enamel. The teeth withpits are the main clinical manifestation of enamel fluorosis. Fluoride may affect ameloblastsby reduced proteolytic activity. Excessive fluorine ions may also cause the oxidative stressreaction and induce cell apoptosis. Fluoride ions can stimulate endoplasmic reticulum stressresponse and affect the synthesis and secretion of proteins. Excess fluoride can also affectthe pH value that cause the amelogenin’s structure and function changes. However, themechanism by which the fluoride to influence intracellular pH and the ways of fluoride passthrough cell membrane are not unclear.We chose ameloblast-like cell lines (LS8cell lines) and treated with excess fluoride, thenew chloride fluorescent probe MQAE was used to test the changes of intracellular chlorideconcentration. After being treated with2mM NaF for half an hour, the average green fluorescence intensity of experimental group cells was4times higher than that of the controlgroup(P<0.01); then MQAE and LysoSensor DND-167were both used in two groups,thecells were taken images at live cell workstation for30min. The results showed that bluefluorescence and green fluorescent of control group become weaker and two kindsfluorescence intensity of experimental group were increased. These results manifested thatthe chloride concentration and intracellular pH was inversely proportional in normal LS8cells, with the reduction of chloride concentration the intracellular pH elevated; whilst in thehigh fluoride treated cells, the higher concentration of chloride ions in the cells the lowerintracellular pH had. Fluoride may change the intracellular pH value by affecting theintracellular chloride concentration.Now there is no obvious evidence to prove the existence of fluoride channel. It isgenerally accepted that fluoride pass through cell membrane via chloride channels, our teamfound that ClC-1, ClC-3, ClC-6and ClC-7may be the fluorine sensitive chloride ionchannels. In order to verify the further mechanism, we used gene transfection to silence thefour genes in ameloblast, and then applied excessive fluorine to the cell. MQAE was used todetect the concentration of the chloride ions. It is found that the intracellular fluorescenceintensity was increased with the increase of extracellular fluorine ion concentration. Excessfluoride can competitively inhibited the extracellular chloride ions into the cell. Instead,when the gene Clcn1Clcn3Clcn6and Clcn7was knocked out, the increase of fluorine ionsdid not affect the concentration of intracellular chloride ions. Thus fluorine ion cannot entercells through these channels and compete with the chloride ion.At the same time we used fluoride probe Chemodosimeter1to test intracellular fluorideconcentration of RAW264.7cell lines between two groups, in which Clcn1Clcn3Clcn6andClcn7genes were knocked down and the control cells. The fluorescence intensity ofChemodosimeter1will be increased with the increase of fluoride concentration. It was foundthat the fluorescence intensity was increased with extracellular fluorine ion concentration incontrol cells. When the four genes were knocked down, extracellular fluorine ions increasedid not affect the intracellular concentration of fluoride ions, which is consistent withexperiment two.In the study we found that the fluorine ions can causes cell acidification by interferingwith the intracellular chloride ion concentration and select several type fluoride-sensitive ClC chloride ion channels and verified that ClC-1, ClC-3, ClC-6and ClC-7are the channelwhich fluorine ions across the plasma membrane. This study is first time to research fluorideion transmembrane transport mechanism from the point on the CLC chloride channel of theview of cell physiology; it plays an important role on elaborated the biological role of thechloride channel.