The Relationship Between The Activation Of Volume-regulated Chloride Channel And The Intracellular Ca²⁺

Volume-regulated chloride channel（VRCC）is a member of the chloride channels,which is broadly expressed and plays a key role in maintaining cell volume and homeostasis.VRCC is also involved in apoptosis and other pathophyslogical processes.Over the years,there have been great debates about the molecular basis of VRCC,and recently,LRRC8 A and other LRCC8 members are identified to be responsible for the formation of VRCC.However,there are also evidence suggesting that TMEM16A（ANO1）and bestrophin,which are the basis of Ca²⁺ activated chloride channels（CaCCs）,also participate in formation of VRCC.Ca²⁺ is an important second messenger,and participates in many cell functions.It has been found that cell swelling is accompanied with an increase of intra-cellular Ca²⁺.Accumulating evidence denmonstrate that Ca²⁺（through a nanodomain mechanism）is possibly an important mechanism involved in the activation of VRCC.This study is aimed to investigate the mechanism of VRCC activation.The first part of the study mainly study the relative contributions of LRRC8 A and ANO1 to VRCC;and the second part mainly study the role of intra-cellular Ca²⁺ in VRCC activation.Part 1 The contributions of LRRC8 A and ANO1 to VRCC.Objective: To compare the relative contributions of LRRC8 A and ANO1 to VRCC in HEK293 cells.Methods:1 Whole cell patch clamp was used to record VRCC in HEK293 cells which was voltage clamped at – 60 mV;the VRCC was activated by either intra-cellular hypertonicity or extra-cellular hypotonicity.2 Assessing the effects of deleting LRRC8 A or ANO1 by CRISPR/Cas 9 technique on VRCC currents.3 Assessing the effects of overexpressing LRRC8 A or ANO1 on VRCC currents.Results:1 The current density of VRCC in HEK293 cells induced by intra-cellular hypertonicity or by extra-cellular hypotonicity was 62.33 ± 3.98 pA/pF（n = 22）,and 59.11 ± 6.20 pA/pF（n = 18,P > 0.05）,respectively.2 When LRRC8 A in HEK293 cells was deleted（LRRC8A-KO）,the VRCC current density decreased from 62.33 ± 3.98 p A/p F to 3.10 ± 0.68 pA/pF（n = 11,** P < 0.01）;and when ANO1 was deleted（ANO1-KO）,the VRCC current was decreased from 62.33 ± 3.98 pA/pF to 49.42 ± 8.36 pA/pF（n = 17,* P < 0.05）.3 VRCC current was rescued by overexpression of LRRC8 A in LRRC8A-KO cells with the current density of 53.34 ± 20.20 pA/pF（n = 7,P > 0.05）;overexpression of ANO1 although also increased the VRCC current density（9.15 ± 1.91 p A/pF,n = 10,*** P < 0.001）,it did not revover the VRCC cuurent to the control HEK cell level（62.33 ± 3.98 p A/pF,n = 22）;and the VRCC current mediated by ANO1 has the current kinetics similar to thoese of the currents from Ca²⁺ activated chloride channel（CaCCs）.Conclusion:1 In HEK cells,the current density and kinetics of VRCC currents indeuced either by intra-hypertonicity or extra-hypotonicity are similar,both with characteristics of outwardly rectification and inactivation at positive voltages.2 Both LRRC8 A and ANO1 participate in forming VRCC in HEK cells,with LRRC8 A to be the bigger contributor.Part 2 The involvement of intra-cellular Ca²⁺ in VRCC activationObjective: To evaluate the relationship between intra-cellular Ca²⁺ and VRCC activation,to compare the intracellular Ca²⁺ changes and VRCC currents induced by extra-cellular hypotonicity,and by serum and ATP in an isotonic extra-cellular solution;to explore the sourse of increased intracellular Ca²⁺ induced by cell swelling.Methods:1 Whole cell patch clamp was used to record VRCC in a holding potential of-60 mV,the effects of intra-cellular Ca²⁺ chelators BAPTA and EGTA on VRCC currents were evaluated.2 The effects of extraxellular on Ca²⁺ on VRCC were studied.3 The effects of extra-cellular hypotonicity,and the effects of serum and ATP in an isotonic extra-cellular solution on intracellular Ca²⁺ were studied.4 VRCC currents induced by serum and ATP in an isotonic extra-cellular solution were recorded and compared.5 The effects of Ryanodine receptor agonist dantrolene or antagonist caffeine on VRCC currents were recorded and compared.6 The effects of deleting phospholipase PLCγ1 and PLCβ4 with CRISPR/Cas 9 technology on VRCC currents were studied.7 The effects of extra-cellular hypotonicity on membrane PIP2 were studied.8 The effects of membrane lipid-raft modifier M-βCD on VRCC currents induced by extra-cellular hypotonicity,and induced by serum and ATP in an isotonic extra-cellular solution were studied.Results:1 Fast chelation of intracellular Ca²⁺ inhibited VRCC.The VRCC currents induced by intracellualr hypertonicity were decreased significantly by intra-cellular BAPTA,from 76.11 ± 4.10 pA/p F（n = 12）to 15.72 ± 3.63 pA/pF（n = 9,***P < 0.001）,while EGTA did not affect siginicantly the VRCC（63.99 ± 6.09 pA/pF,n = 16,P > 0.05）.Similarly the VRCC currents induced by extracellular hypotonicity were also inhibited by intracellular BAPTA,the current density was decreasd from 56.36 ± 10.94 pA/pF to 2.88 ± 0.51 pA/pF（n = 8,*** P < 0.01）.2 The VRCC current density in the absence of extracellular Ca²⁺ was 74.28 ± 11.44 pA/p F（n = 12）,which was not significantly diffent from control,about 62.33 ± 3.98 pA/pF（n = 22,P > 0.05）.3 Extra-cellular hypotonicity,serum and ATP in an isotonic extra-cellular solution all induced intra-cellular Ca²⁺ transient,although the characteristics of these Ca²⁺ transient were different.Deletion of LRRC8 A did not affect the intra-cellular Ca²⁺ concerntration;removal of extra-cellular Ca²⁺ affected the ATP-induced Ca²⁺ transient significantly.4 In isotonic condition,serum induced a transient activation of VRCC,with a current density of 6.88 ± 1.58 p A/p F（n = 12）;ATP could not induce VRCC.5 VRCC activation was affected when function of Ryanodine recptor was modified.The current density of VRCC decreased from 71.61 ± 4.89 pA/pF to 25.51 ± 1.97 p A/p F（n = 16,*** P < 0.001）after incubation with dantrolene,the RyR antagonist,and only two cell showed small VRCC current density of 4 p A/pF and 25 pA/pF respectively out of all 11 cells when treated with RyR agonist caffeine.6 PLC was not involved in the activation of VRCC.The VRCC current density was 67.05 ± 9.53 p A/pF（n = 14）and 71.61 ± 8.81 pA/pF（n = 9）,respectively in HEK cells where PLCγ1 or PLCβ4 was deleted,which was not significantly different from the control cells,about 79.95 ± 3.85 pA/pF（n = 10,P > 0.05）.7 Extracellular hypotonicity did not trigger the hydrolysis of membrane PIP2.8 Destroying the lipid raft with M-βCD did not affect the activation of VRCC induced by extracellular hypotonicity or serum in an isotonic condition.Conclusion:1 Cell swelling induces an increase of intra-cellular Ca²⁺,which in the form of nanodomain participates in the activation of VRCC.The increased intra-cellular Ca²⁺ induced when VRCC is activated derives from the intracellular Ca²⁺ store,but not from extra-cellular Ca²⁺ influx.PIP2 pathway and PLC pathway probably do not participate in the activation of VRCC,and are probably not the source of nanodomain Ca²⁺.2 Similar to extra-cellular hypotonicity,serum and ATP in an isotonic condition also induce the intra-cellular Ca²⁺ transient.However serum can and ATP can not induce activation of VRCC,indicating that Ca²⁺ alone can not activate VRCC,and furthermore,the cell volume swelling is not an essential factor in activation of VRCC.3 Structure of lipid-raft is not an essential part of of VRCC activation.