| The volume-sensitive chloride channel?full name: volume-sensitive outward rectification chloride channel?also known as volume-regulated anion channel is abundantly distributed in mammalian tissues and cells.It is involved in many pathological and physiological processes such as regulating cell volume,cell proliferation and differentiation,immune response,cell membrane potential,intracellular p H and cell apoptosis.Otherwise it is also involved in the occurrence and development of a variety of cardiovascular system diseases.For example,blocking VSOR Cl-channel can extend the action potential time course and myocardial refractory period to prevent malignant arrhythmia,and can reduce ischemia reperfusion injury,and inhibit cardiac hypertrophy,and reduce myocardial infarction scope,finally restore the basic functions of the heart.So the intervention and regulation of VSOR Cl-channel functions can become a target to protect myocardium.In recent years,due to the continuous study of the channel,LRRC8 A as known as the encoding protein of VSOR Cl-channel discovered in 2014 laid a solid foundation for further study of VSOR Cl-channel.Recent studies have shown that VSOR Cl-channels are not directly activated by mechanical stress stimulation,but rather involve the sensitivity of ionic strength and cell membrane tension,which involves the actin cytoskeleton,membrane cell membrane protein,cell membrane lipid composition and G-protein.Moreover,the study found that not only the ion channel itself,but also the combination of abnormal and/or functional changes in protein structures can lead to a series of pathological changes.Caveolin,which has a membrane reserve function,is the most common ion channel binding protein on the cell membrane.Studies have shown that caveolin can form complexes with cationic channels such as K+,Na+,Ca2+ and other complexes.Caveolin structure changes and functional abnormalities can cause the above-mentioned abnormalities of these ion channels,and even cause systemic diseases and ion channel disease.However,there are only a few cases of reports about the relationship between caveolin and anion channels,and there is none reports about the relationship between caveolin and VSOR Cl-channel and its related mechanism.Therefore,this study intends to:1)To investigate the localization of VSOR Cl-channel encoding protein LRRC8 A on the cell membrane and its relationship with caveolin;2)To further explain the effect of caveolin on the VSOR Cl-channel function,whether it controls the activation of the VSOR Cl-channel,and how can it play a regulatory role?Objectives: 1.To explore the localization of Cav-1/3 and LRRC8 A on cardiomyocytes and their relationship 2.To explore whether Cav-1/3 has an effect on VSOR Cl-channel function 3.To investigate the effect of Cav-1/3 on the expression of VSOR Cl-encoded protein LRRC8AMethods: 1.The experimental group: isotonic group,hypotonic group,and the experimental group?hypotonic + DIDS / hypotonic+ M?CD?,Cav-1/3 siRNA+isotonic,Cav-1/3 siRNA + hypotonic.2.Culture of primary neonatal rat cardiac myocytes: conventional isolation of rat heart,cutting,digestion,differential adherence and culture 3.The location of LRRC8 A,Cav-1 and Cav-3 was detected by fluorescencestaining.4.The caveolin-rich fragment was obtained by sucrose density ultra-centrifugation after a number of primary cultured neonatal cardiomyocytes were cleavage by ultrasound.5.Western blot was used to detect target protein LRRC8 A,Cav-1,and Cav-3.6.Co-Immunoprecipitation was used to detect the interaction between Cav-1 and LRRC8 A,Cav-3 and LRRC8 A.7.The primary cultured neonatal rat cardiomyocytes were treated with hypotonic solution,hypotonic solution with DIDS,and hypotonic solution with M?CD in orde to record ICl,vol using full-cell recording mode by patch-clamp technique.8.Subcultured cardiomyocytes?H9C2?being transfected with siRNA silencing Cav-1 or Cav-3 for 48-72 h were re-stick after trypsin digestion for about 1h.The current of ICl,vol was record by patch clamp,and the expression of LRRC8 A with the changes of Cav-1 expression tested by western blot.9.Flow cytometry was used to detect the changes of the cell volume under isotonic solution,hypotonic solution,hypotonic + M?CD solution on subcultured H9C2 cardiomyocytes and plot the graph.10.MQAE was used to detect the fluorescence density changes of the cardiomyocytes treatmented with isotonic,hypotonic,hypotonic+DIDS and hypotonic+M?CD under inverted microscope and microplate reader.11.Cav-1 gene was silenced or overexpressed into the subcultured cardiomyocyte line?H9C2?.The expression of LRRC8 A membrane protein was detected by immunoblotting with the expression of Cav-1 protein.Results: 1.In the primary cultured cardiomyocytes,the Cav-1/3 and LRRC8 A were labeled with different fluorescent secondary antibodies.Fluorescent inverted microscope showed Cav-1/3?red?,LRRC8A?green?,consistent distribution?yellow?,the results showed that there is a common relationship between Cav-1/3 and LRRC8 A.2.The primary cultured cardiomyocytes were harvested by sucrose density ultracentrifugation,and 1-12 parts of different cell pellets according to the results of ultrasonic centrifugal stratification.After centrifugation and protein extraction,the western blot showed that Cav-1,Cav-3 and LRRC8 A are distributed in the same proportion of membrane cell enrichment zone and form an immune coprecipitation complex,suggesting that both Cav-1/3 are in direct contact with LRRC8 A.3.ICl,Vol current can be recorded?220 Osmol/kg H2O?by hypotonic perfusion in the primary cultured cardiomyocytes,which shows a high voltage,time-dependent loss when the depolarization voltage reaches to +80 m V.The current density at + 100 m V was 98.45 ± 1.76 PA/p F?n = 5?,which can be blocked by DIDS at +40 m V,+60 m V,+80 m V and + 100 m V.The I/V curve indicated that the current exhibits outward rectification.The inhibition rate at +100m V was 90.22 ± 11.50%?P <0.01 vs HYPO,n = 5?.4.After recording the positive ICl,vol reduced by hypotonic perfusion in the primary cultured cardiomyocytes,then hypotonic solution containing 10mmol/L M?CD was added,the current was also blocked at +40 m V,60 m V,+80m V and +100m V.The current density at + 100 m V was 36.57 ± 10.46 PA/p F,the inhibition rate was 91.86 ± 12.70%?P < 0.01 vs HYPO,n = 3?.5.The cells were pre-incubated with hypotonic perfusion containing 2.5mmol /L M?CD for half an hour and replaced with hypotonic perfusion containing the same concentration of M?CD.ICl,Vol significantly decreased at +40m V,+60m V,+80m V and +100m V,showing a voltage-dependent blockade.The current density at + 100 m V was 25.95 ± 4.75 p A / p F,and the inhibition rate was 89.19 ± 8.35%?P <0.01 vs HYPO,n = 4?.6.After treatment with Cav-1 siRNA and Cav-3 siRNA for 72 hours,ICl,vol was not reduced in the Cav-1 siRNA group cells perfused by hypotonic solution.The current density at + 100 m V was 28.53±7.04 p A/p F,and the inhibition rate was 78.27±7.93% ?P<0.01 vs HYPO,n=4?.While positive ICl,vol was also recorded in the Cav-3 siRNA group cells perfused by hypotonic solution.The current density at + 100 m V was 77.85± 3.04 p A/p F,and there is no significant difference compared to HYPO?P>0.05 vs HYPO,n=4?.7.The cell volume of H9C2 cells was measured by flow cytometry after treatment with isotonic,hypotonic,and hypotonic + M?CD for 1 h.The results showed that the cell volume did not increase in the hypotonic+ M?CD group compared to hypotonic group.It suggested that caveolin may play a role in regulating cell volume by intervening in the VSPR Cl-channel function.8.We used MQAE to detect intracellular chloride ion concentration.The results showed that the fluorescence intensity of the hypotonic group was the strongest compared to hypotonic+DIDS and hypotonic+ M?CD?P<0.01,n=6?.But the differences between the two groups?hypotonic+DIDS and hypotonic+ M?CD?were not significant?P>0.05,n=6?.9.The expression of LRRC8 A membrane protein decreased after treatment with Cav-1 siRNA,and there were significant difference in statistics?P<0.05,n=6?.The expression of LRRC8 A membrane protein was not affected by over expression of Cav-1,and the difference between them was not significant?P> 0.05,n=6?.Conclusions: 1.Cav-1/3 and LRRC8 A are located in the caveolae of the cardiomyocytes,showing a co-localization relationship.2.Destruction and interference with caveolin can significantly reduce the volume of sensitive chlorine current under hypotonic stimulation,preventing the cell volume increases in myocardial cells.3.Cav-1 regulates LRRC8 A,and the expression level of Cav-1 regulates the function of LRRC8 A protein. |
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