Characterization And Mechanism For Pharmacological And Cell Volume Regulation Of Cl~- Channels

The calcium activated chloride channels?CaCCs?are anionic channels with both calcium and voltage dependence.CaCCs play a variety of physiological roles in many organs and tissues,including sensory transmission,regulation of neuronal and cardiac excitability,regulation of vascular tone and transepithelial Cl-secretion.A number of candidate proteins have been proposed to form CaCC,but only two families,the bestrophins and the TMEM16 proteins?ANO1?,recapitulate properties of native CaCC in expression systems.When expressed in the expression systems,both ANO1 and Bestrophin1 show the characteristics of endogenous CaCC: Cl-currents with Ca²⁺ and voltage dependency.Both channels are sensitive to broad-spectrum Cl-channel blockers,which make it difficult to reveal molecular identity of endogenous CaCCs.Studies of endogenous CaCCs are hindered by the lack of specific pharmacological modulators as most Clchannel modulators lack selectivity and a systematic comparison of the effects of these modulators on ANO1 and bestrophin is missing.Profiling of different modulators of ANO1 and Bestrophin1 will benefit our usage of these modulators in studying CaCC and promote development of more specific modulators of CaCC.In the first part of this study,we studied seven Clchannel inhibitors: niflumic acid?NFA?,NPPB,flufenamic acid?FFA?,DIDS,tannic acid,CaCCinh-A01 and T16Ainh-A01 for their effects on the currents ANO1 and Bestrophin1 stably-expressed in CHO cells using patch clamp technique.Volume regulated chloride channel?VRCC?is an anionic channel which plays a very important role in regulating cell volume.VRCC also plays an important role in cell proliferation,differentiation and apoptosis.In addition,VRCC is also suggested to contribute to ischemic injury and anti-cancer drug resistance.However,the study of molecular identity of VRCC is slowly,P-glycoprotein,nucleic acid-sensitive chloride channel protein,voltage-dependent chloride channel ClC-2 and ClC-3,and calcium-activated chloride channel ANO1 and bestrophin were all considered to be involved in the consititution of VRCC.In 2014,two groups reported that LRRC8 family proteins are an essential component of VRCC.The second part of this study focus on the molecular basis and pharmacological characteristics of VRCC in HEK293/CHO cells,with a focus on the role of ANO1 and LRCC8 A.It is still unknown how the VRCC is activated by the increase of cell volume.It has been proposed that intercellular calcium,ionic strength,small G protein RhoA,phosphorylation,tyrosine kinases and reactive oxygen species?ROS?play roles in the activation of VRCC.The third part of this thesis will study the activation mechanism of VRCC in HEK293 A cell and will focus on the roles of intercellular calcium and PLC in the activation of VRCC.Part 1 Characterization of the effects of Cl-channel modulators on ANO1 and Bestrophin1 Ca²⁺ activated Cl-channelsObjective: To investigate the effects of seven Cl-channel modulators on ANO1 and Bestrophin1 Ca²⁺ activated Cl-channels.Methods: 1 Establish the ANO1 and Bestrophin1 stably transfected CHO cell lines.2 Whole-cell patch clamp was used to record ANO1 and Bestrophin1 currents and the efficacy,potency and dynamics of different Clchannel modulators on these two currents were compared.Results: 1 We first established the ANO1 and Bestrophin1 stably-transfected CHO cell lines.Compared with untransfected CHO cells,overexpression of ANO1 and Bestrophin1 not only resulted in prominent membrane expression of these channels,but more importantly produced large intracellular Ca²⁺-dependent currents with voltage dependency and outwardly rectification.2 Among seven inhibitors studied,NFA showed highest selectivity for ANO1?IC50 of 7.40 ± 0.95 ?M?at +80 mV over Bestrophin1?IC50 of 102.19 ± 15.05 ?M?.In contrast,DIDS displayed a reverse selectivity inhibiting Bestrophin1 with IC50 of 3.93 ± 0.73 ?M and ANO1 with IC50 of 548.86 ± 25.57 ?M.CaCCinh-A01 was the most efficacious blocker for both ANO1 and Bestrophin1 channels.T16Ainh-A01 partially inhibited ANO1 currents but had no effect on Bestrophin1 currents.Tannic acid,NPPB and FFA had variable intermediate effects.3 NFA,NPPB and FFA showed dual effects on ANO1 inward current and increased the inward currents of ANO1 at low concentrations?< 100 ?M?and inhibited the currents at high concentrations?> 100 ?M?.NFA,NPPB and FFA markedly slowed the deactivation of ANO1 currents.Conclusions: 1 The ANO1 and Bestrophin1 stably-expressed CHO cell lines were successfully established.2 Among seven inhibitors studied,NFA showed highest selectivity for ANO1 over Bestrophin1.In contrast,DIDS displayed a reverse selectivity inhibiting Bestrophin1 and ANO1.CaCCinh-A01 was the most efficacious blocker for both ANO1 and Bestrophin1 channels.3 NFA,NPPB and FFA showed dual effects on ANO1 inward current and markedly slowed the deactivation of ANO1 currents.Part 2 Involvement of ANO1 in molecular composition of volume regulated chloride channelObjective: To investigate the molecular identity of volume regulated chloride channel?VRCC?and study whether ANO1 is involved in the constitution of VRCC.Methods: 1 Whole-cell patch clamp was used to record the VRCC currents from HEK/CHO or ANO1 stably transfected HEK/CHO cells with 420 mOsm CsCl intracellular solution and 320 mOsm NaCl bath solution.2 The effect of chloride channel inhibitors on VRCC currents was studied.3 The LRRC8 A and ANO1 knockout HEK293 A cell lines were established using CRISPR/Cas9 technique.Results: 1 An outwardly rectifying chloride current?VRCC?was elicited by the hypertonic pipette solution?420 mOsm containing CsCl?in HEK293 cells.The VRCC currents were recorded with a ramp protocol from-100 mV to +100 mV.The current density of the VRCC currents in HEK293 cells were about 64 pA/p F?48 pA/pF in CHO cells?at-60 mV,and the current density of VRCC in ANO1 stably transfected HEK?HEK-mANO1?cells were about 113 pA/pF?63 pA/pF in ANO1 stably transfected CHO cells?,which was significant larger than that in the control HEK cells.When recorded using a step voltage protocol,the activated VRCC currents in HEK-mANO1 cells showed the properties of slow time-dependent activation and non-deactivation,which was different from the slow deactivation of the VRCC currents recorded in the control HEK cells.2 NFA?100 ?M?which was showed in the first part as a potent inhibitor of ANO1 currents,partly inhibited the VRCC currents recorded in HEK-mANO1 cells;the currents inhibited by NFA displayed the slow activation and non-deactivation,consistent with the properties of ANO1 currents.DCPIB?10 ?M?,which was suggested to be selective inhibitor of VRCC currents also partly inhibited VRCC currents recorded in HEK-mANO1 cells;different from NFA,the current DCPIB inhibited displayed slow deactivation,possibly suggestive of a LRRC current.3 VRCC currents in the control HEK cells were markly reduced when ANO1 was knockout.The VRCC currents were almost abolished when LRRC8 A was knockout.However,in these LRRC8 A knockout cells,expression of ANO1 restored the VRCC currents with characteristics of slow activation and non-deactivation.Conclusions: 1 VRCC currents were much larger in ANO1 stably expressed HEK cells compared with that in the control HEK cells.VRCC currents were significantly reduced in the ANO1 knockout cells.2 VRCC currents were abolished in the LRRC8 A knockout HEK cells and were partly restored when ANO1 was transfected in these cells.The results indicate that ANO1 can be activated by the cell volume expansion and could be a constitute of the VRCC.Part 3 Mechanism for the activation of volume regulated chloride channelObjective: To investigate the activation mechanism of VRCC currents in HEK cells.Methods: 1 VRCC currents in HEK293 A cells were recorded using whole-cell patch clamp technique.2 Intracellular Ca²⁺ changes was monitored by LSCM.3 The role of PLC pathway involvement was assessed using the PLC inhibitors or siRNA.4 The membrane PIP2 hydrolysis was monitored using the specific fluorescence dyes tubby-YFP.Results: 1 VRCC currents recorded in HEK293 A cells with the hypertonic intracellular solution showed oscillation.And the current deactivation became slower as the amplitude of currents increased.VRCC currents induced by the hypotonic bath solution showed the same characteristic with current induced by the hypertonic intracellular solution.2 Ca²⁺ oscillation was seen when cells were exposed on hypotonic bath solution and 2 mM EGTA or CdCl2?which were added in the bath solution to reduce extracellular calcium and calcium channels,respectively?did not change Ca²⁺ oscillation or Ca²⁺ rising.3 EGTA added in the hypertonic pipette solution abolished the current oscillation but did not affect the maximal current amplitude.Adding BAPTA in the pipette or pretreating the HEK cells with thapasigagin reduced the VRCC currents significantly.4 A transicent VRCC-like current were induced by 3‰ rat serum and the currents were almost totally abolished by DCPIB and CaCCinh-A01.The serum also induced a Ca²⁺ transcient increase in HEK cells.5 U73122?5 ?M?,an inhibitor of PLC,but not the inactive analog U73343 inhibited the VRCC.6 The tubby-YFP indicated membrane PIP2 was significantly decreased when the cells were subjected to the hypotonic bath solution stimulation.7 The VRCC currents were decreased significantly by IP3 receptor inhibitor Xe-C but not by PKC blocker Bis-1.Adding GDP-?s?500 ?M?in the pipette solution did not affect the development of the VRCC.8 The VRCC currents were decreased significantly when HEK293 A cells were transfected with siRNAs against PLC?4,PLC?1 or PLC?3 siRNA but not by PLC?3 and PLC?1.Conclusions: 1 The VRCC currents recorded in HEK293 A cells oscillated and the current dynamic characteristics were different at the peak and the trough of the current amplitudes.2 Activation of VRCC in HEK293 A cells was dependent on the intracellular calcium,especially a local calcium rising.3 Activation of VRCC was dependent on PLC activation and the isoforms of PLC?1 and PLC?4 and PLC?3 were possibly involved.4 G protein and PKC did not participate in the activation of VRCC.