Identification Of Phospholipase C Isoforms Involved In The Activation Of Volume-regulated Anion Channel

VRAC（Volume Regulatory Anion Channel,VRAC）are anion channels that are activated when the cell volume are enlarged.VRAC are wildely expressed in human tissues and play important physiological functions.VRAC are activated to generate a protective process called RVD（regulatory Volume decrease,RVD）when cell volume is enlarged,to realize a protection role for cell volume.VRAC is involved in many physiological and pathlogical processes including cell migration,proliferation,apoptosis and obesity.Recently,two laboratories have found that LRRC8A homologous oligomers are the molecular basis of VRAC,but its activation mechanism is still not clear.It has been indicated that intracellular Ca²⁺is involved in the activation of VRAC and the results of previous studies in our laboratory have confirmed that the activation of VRAC is affected by modulation of intracellular Ca²⁺.It also has been indicated that cell swelling is companied with a rising of intracellular Ca²⁺.It is well known that activation of phospholipase C（PLC）will induce a release and rising of intracellular Ca²⁺.With these evidence,we speculate that the cellular PLC singal pathway may participate in the activation of VRAC.This study focuses on the role of PLC pathway in activation of VRAC.The study is divided into two parts:the first part mainly deals with the establishment of a YFP stable cell line,and to verify the validity of this cell line for further use in the evaluation of PLC isoforms in the activation of VRAC,using confocal,patch clamp and PLIPR techniques.In the second part of the study,high-throughput screening（HTS）using YFP cell line and FLIPR was used to observe the effect of siRNA against different PLC isoforms on the activation of VRAC,and the results was further validated using the electrophysiological patch clamp and qPCR method,to explore the contribution of different PLC subtypes in activation of VRAC.Part 1 Establishment of YFP-H148Q-I152L stable cell line and platformfor measurement of VRAC functionObjective:To obtain the YFP-H148Q-I152L-stable-transfected cell line,and to verify the cell line with methods of measurement of VRAC function including the HTS（high throughout screening）platform.Methods:1 YFP-H148Q-I152L plasmid was prepared and transfected into blank HEK293A cells.The HEK293A cells expressing YFP（YFP-HEK293）were obtained.2 The death curve of HEK293A cells was established with use of the blasticidin S,and the minimum lethal concentration was determined to be 15μg/mL.The single YFP-HEK293A cell was obtained by infinite dilution method.The YFP-HEK293A cells were cultured in medium containing 15μg/mL blasticidin S for about 2 weeks.3 The function of YFP-HEK293A cells was measured with method of confocal imaging,FLIPR high throughput fluorescence system and patch clamp electrophysiology.Results:1.In confocal experiments,the YFP-HEK293A cell line show green fluorescence when cxcited by light at 488 nm.90.46%±8.34 of the YFP–HEK293A cells from different batch（N=7）was YFP positive.On the other hand,23.6%±11.56（N=6）of the HEK293A cells was YFP positive when the cells were transfected transiently with YFP–H148Q-I152L plasmid.The intracellular fluorescence intensity of both types of YFP-HEK293A cells were bleached greatly by influxed I^-when the VRAC of YFP-HEK293A cells were activated by the hypotonic extracellular solution which contained NaI.And the 10μM DCPIB（a VRAC blocker）significantly blocked this process.2.In electrophysiological experiments,when the VRAC were activated by the hypotonic extracellular solution,the current density of the YFP–HEK293A cell line under-60 mV was 118.71±14.56 pA/pF（n=9）,while the current density of the blank HEK293A was 103.24±11.38 pA/pF（n=13）;the current density was not statistically significant difference between these two types of cells.In both cells,the VRAC currents were inhibited by 10μM DCPIB.3.In FLIPR fluorescence high-throughput experiments,the fluorescence intensity of YFP–HEK293A cell and the YFP transiently transfected HEK293A cells were bleached by 90.68%±4.92（n=34）and 92.13%±3.35（n=25）,respectively,by the influxed I^-in the hypotonic extracellular solution;no difference was found between these two types of HEK293A cells.Bleaching of YFP fluorescence intensity by I^-was significantly reduced in YFP-293A cells by 7μM U73122（PLC blocker）or 10μM DCPIB（both incubated for 20 minutes）;reduction of fluorescence intensity reduction in these two cases was 54.43%±7.68（n=21,*P<0.05）and 25.61%±3.93（n=18,***P<0.001）,respective,as compared to a reduction of 93.34%±5.45（n=12）in the absence of the blockers.Conclusions:The stable YFP-HEK293A cell line was successfully established.When characterized with confocal,patch clamp and FLIPR techniques,the YFP-HEK293A cells were fund to be similar to the untransfected blank HEK293A cells,including similar VRAC currents density.VRAC was fund to be activated by hypotonic extracellular solution around 5min.which can be blocked by the VRAC’s blocker,DCPIB.The YFP-HEK293A cells lines can be used on FLIPR system for high throughput screening study.Part 2 Role of different PLC isoform in the acvtiavtion of VRACObjective:To evaluate the contribution of different PLC subtypes to the activation of VRAC using gene silencing method;qPCR method was used to determine the knockdown efficiency of siRNA against PLC subtypes.Methods:1 The siRNA targeting different PLC subtypes was transiently transfected into the YFP-HEK293A cells in order to reduce the expression of the PLC subtypes.2 The knock-out efficiency of each PLC subtype was examined using qPCR technology.3 FLIPR high-throughput fluorescence detection system was used to screen for PLC subtypes which are involved in the activation of VRAC.4 Electrophysiological patch clamp experiments were performed to verify the results from FLIPR experiments.Results:1.The PLC inhibitor U73122 blocked the activation of VRAC measured using patch clamp,FLIPR and confocal nethods,indicating that PLC pathway was possibly involved in the activation of VRAC.2.The results of qPCR showed that the corresponding siRNA againt PLCβ3,PLCβ1,PLCζ1,PLCδ4,PLCδ3,PLCβ4,PLCγ1,PLCλ2,PLCη1 and PLCχδ1 subtypes,reduced the expression of these PLC subtypes by 54.12%,61.26%,67.86%,57.21%,51.43%,59.56%,49.67%,71.24%,65.67%and 55.12%,respectively.3.In the FLIPR high-throughput fluorescence detection experiment,the reduction of fluorescence intensity after PLCγ1,PLCβ3,PLCβ1,PLCδ3,PLCδ4,PLCχδ1 and PLCλ2 siRNA treatment was 37.66%±4.68（n=18,*P<0.05）,38.12%±6.89（n=18,*P<0.05）,51.68%±9.56（n=17,*P<0.05）,33.57%±5.46（n=17,**P<0.01）,22.51%±4.65（n=18,*P<0.05）,41.78%±11.54（n=12,*P<0.05）and 44.51%±8.86（n=17,*P<0.05）,respectively,which was significantly smaller than no-siRNA treated YFP-HEK293A cells,87.63%±8.54（n=48）.Whereas when silencing PLCβ4,PLCη1,PLCζ1,PLCε1,PLCχδ2,PLCβ2,PLCη2 and PLCλ1 the fluorescence intensity changed to 84.78%±11.23（n=17）,80.24%±13.45（n=18）,80.6%±13.53（n=12）,86.74±10.69（n=18）,81.33%±11.35（n=18）,86.74%±9.69（n=12）,81.67±9.79（n=17）and 84.33%±11.43（n=12）,respectively,compared to control group,the difference was not statistically significant.4.The patch clamp experiments were carried on the YFP-HEK293A cells treated with siRNA against PLCγ1,PLCβ3,PLCβ1,PLCδ3,PLCδ4,PLCχδ1and PLCλ2 subtypes,and the current density of VRAC in these cells was22.54±4.32 pA/pF（n=13,*P<0.05）,29.68±5.62 pA/pF（n=12,**P<0.01）,27.24±6.23 pA/pF（n=14,**P<0.01）,35.27±8.62 pA/pF（n=13,*P<0.05）,21.65±3.38 p A/p F（n=17,***P<0.001）,20.35±7.24 pA/pF（n=13,**P<0.01）and 32.47±7.64 pA/pF（n=11,**P<0.01）,respectively,whereas the current densities of control cells,VRAC in non-treated scrambled siRNA-treated cells,the cells treated with siRNA against PLCζ1,PLCβ4,PLCη1 were 128.65±21.34 pA/pF（n=11）,145.32±17.88 pA/pF（n=7）,132.92±28.75 pA/pF（n=9）,113.93±19.85 pA/pF（n=8）and 98.49±19.75 pA/pF（n=9）,respectively.The difference was not statistically significant.Conclusion:Seven PLC subtypes（PLCγ1,PLCβ3,PLCβ1,PLCδ3,PLCδ4,PLCχδ1 and PLCλ2）are possibly participated in the regulation of VRAC activation.