Biophysical characterization of chloride intracellular channel 6 (CLIC6)

Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K+) solutions, we determined that CLIC6 is more permeable to chloride-(Cl−) as compared to bromide-(Br−), fluoride-(F−), and K+ ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 μM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl− channel. Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K+) solutions, we determined that CLIC6 is more permeable to chloride-(Cl−) as compared to bromide-(Br−), fluoride-(F−), and K+ ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 μM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl− channel. The chloride channels are present in all the cellular membranes and are tightly regulated by various stimuli including voltage, pH, volume, ligands, pressure, and intracellular messengers (1Verkman A.S. Galietta L.J.V. Chloride transport modulators as drug candidates.Am. J. Physiol. Cell Physiol. 2021; 321: C932-C946Crossref PubMed Google Scholar, 2Jentsch T.J. Stein V. Weinreich F. Zdebik A.A. Molecular structure and physiological function of chloride channels.Physiol. Rev. 2002; 82: 503-568Crossref PubMed Scopus (1080) Google Scholar). These channels are preferentially permeable to chloride and often allow other smaller anions as well as cations to pass through them (3Duran C. Thompson C.H. Xiao Q. Hartzell H.C. Chloride channels: often enigmatic, rarely predictable.Annu. Rev. Physiol. 2010; 72: 95-121Crossref PubMed Scopus (274) Google Scholar). In cells, physiologically chloride channels play a vital role in regulating their homeostasis, including stabilization of cell membrane potential, transepithelial transport, maintenance of intracellular pH, cell proliferation, fluid secretion, and regulation of cell volume (2Jentsch T.J. Stein V. Weinreich F. Zdebik A.A. Molecular structure and physiological function of chloride channels.Physiol. Rev. 2002; 82: 503-568Crossref PubMed Scopus (1080) Google Scholar, 4Gururaja Rao S. Patel N.J. Singh H. Intracellular chloride channels: novel biomarkers in Diseases.Front. Physiol. 2020; 11: 96Crossref PubMed Scopus (63) Google Scholar). Several families of chloride channels have been identified over the past 3 decades. Members of the recently characterized chloride intracellular channel (CLIC) protein family have been reported in intracellular organelles (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar). CLICs have six paralogs, CLIC1-6, and they exist in soluble as well as membrane forms (6Singh H. Two decades with dimorphic chloride intracellular channels (CLICs).FEBS Lett. 2010; 584: 2112-2121Crossref PubMed Scopus (62) Google Scholar). Their dysregulation is associated with cancer (7Malik M. 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Intracellular chloride channels are present in membranes they are involved in the regulation of pH, volume, and of organelles Rao S. Patel N.J. Singh H. Intracellular chloride channels: novel biomarkers in Diseases.Front. Physiol. 2020; 11: 96Crossref PubMed Scopus (63) Google Scholar). family are found in the K. et and of a chloride channel of cell PubMed Scopus Google D. S. S. D. et localizes to membranes and PubMed Scopus Google Scholar, D. Rao S. J. K. et of chloride intracellular channel PubMed Scopus Google Scholar, et chloride channel is by and in the to 2002; PubMed Scopus Google D. Rao J. K. et of and and of CLICs in of PubMed Scopus Google M. J. et at the of with a novel chloride intracellular channel (CLIC) family 2002; PubMed Scopus Google and membranes J. A. et and to from and cancer 2012; PubMed Scopus Google and as a soluble protein in the J. and of Cell PubMed Scopus Google Scholar). CLIC6, the most family member was identified in and (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar, D. Singh H. channels of Pharmacol. PubMed Scopus Google Scholar, M. M. S. D. M. et of a novel member of the CLIC6, to PubMed Scopus Google Scholar, F. F. J. CLIC6, a member of the intracellular chloride channel with PubMed Scopus Google Scholar, A. M. M. of CLIC6 by and 2018; PubMed Scopus Google Scholar, Molecular and of a novel chloride intracellular expressed in PubMed Scopus Google Scholar). CLIC6 is present in the in and is the known of proteins M. M. S. D. M. et of a novel member of the CLIC6, to PubMed Scopus Google Scholar, A. M. M. of CLIC6 by and 2018; PubMed Scopus Google Scholar, Molecular and of a novel chloride intracellular expressed in PubMed Scopus Google Scholar). proteins present in the are and also that channels have and properties in cells. CLIC6 the with other proteins as well as the (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar, Rao S. Ponnalagu D. S. Singh H. S. et and of a of chloride Intracellular (CLIC) PubMed Scopus Google Scholar). the was discovered over decades M. M. S. D. M. et of a novel member of the CLIC6, to PubMed Scopus Google Scholar, F. F. J. CLIC6, a member of the intracellular chloride channel with PubMed Scopus Google of the of CLIC6 to channel is recently associated CLIC6 with L. Li J. et novel and of PubMed Scopus Google lung function S. K. et to J. PubMed Scopus Google associated with A. J. M. K. C. et is in and regulated by in 2020; PubMed Scopus Google and in cancer D. K. S. J. K. et associated with in the of cancer PubMed Scopus Google Scholar). CLIC6 also known as in the cell the of Cl− from cells. The Cl− was Cl− was from the CLIC6 in cellular Cl− Molecular and of a novel chloride intracellular expressed in PubMed Scopus Google Scholar). In of CLIC6 with in cell to present Cl− the of CLIC6 to channel F. F. J. CLIC6, a member of the intracellular chloride channel with PubMed Scopus Google Scholar). in cell ectopic CLIC6 to to the plasma membrane of and cells, and Cl− were by Cl− as and F. F. J. CLIC6, a member of the intracellular chloride channel with PubMed Scopus Google Scholar, Molecular and of a novel chloride intracellular expressed in PubMed Scopus Google Scholar). In this we to characterize CLIC6 and to channels in cell proteins are known to channels ectopic in cell S. S. L. L. et function is of by PubMed Scopus Google Scholar, S. et in the transmembrane of the channel biophysical PubMed Scopus Google and Rao S. Ponnalagu D. S. Singh H. S. et and of a of chloride Intracellular (CLIC) PubMed Scopus Google Scholar, et structure of the soluble of the chloride channel protein J. PubMed Scopus Google Scholar, H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar, H. and transmembrane PubMed Scopus Google Scholar, H. of intracellular and regulation by J. PubMed Scopus Google we CLIC6 to channel. On ectopic in HEK-293 cells, we found that CLIC6 is channel that is to pH and redox that CLIC6 is abundant in lung and as compared to other we the properties of CLIC6 in mouse lung epithelial cells. the that CLIC6 forms a channel that preferentially chloride over other ectopic of CLIC6 in cell in to the plasma membrane F. F. J. CLIC6, a member of the intracellular chloride channel with PubMed Scopus Google Scholar, A. M. M. of CLIC6 by and 2018; PubMed Scopus Google we CLIC6 in HEK-293 CLIC6 was with N terminus and the of the protein was in HEK-293 by HEK-293 were also with in ectopic in HEK-293 cells, CLIC6 is expressed the plasma membrane it with the the plasma To the channel we a whole-cell patch-clamp HEK-293 with CLIC6 and in We a current at C and CLIC6 in HEK-293 is The at membrane and to C and The CLIC6 was the as the in the that is with the to chloride in the in IAA-94 is a known of proteins Rao S. Ponnalagu D. S. Singh H. S. et and of a of chloride Intracellular (CLIC) PubMed Scopus Google Scholar, C. A. Q. and of chloride channels from and PubMed Scopus Google Scholar, C. C. Q. and of a channel in the plasma membrane of by J. 11: PubMed Scopus Google Scholar). The of the current at the the of a Cl− from CLIC6 as IAA-94 currents of 10 μM IAA-94 in to a at and The current at was by the addition of IAA-94 we of CLIC6 In HEK-293 CLIC6 were also the Cl− currents were present in HEK-293 cells, they were by IAA-94 C and To determine the of the CLIC6 the was as a function of the and to the CLIC6 as compared to the as by To the at membrane from to were by a to to currents and The current as a function of membrane was by the current and after the addition of 10 μM of the patch-clamp we also HEK-293 with CLIC6 by using patch-clamp with well of the has is to of the well is as all the including and were determined from after the of a are in and be recorded We recorded currents from HEK-293 with CLIC6 We the using a The whole-cell currents at to whole-cell we currents also be by We recorded CLIC6 in the chloride was the and the at and and 10 μM IAA-94 in the In we of the channel in addition to a current The of CLIC6 by and at and the addition of 10 μM IAA-94 We also a of the in was also reported other proteins H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar). demonstrate that CLIC6 a channel that be by proteins are known to The of CLIC6 was by in the The was at a Cl− with the in the and in the we currents in HEK-293 with The channel was C and On with in in the HEK-293 cell with CLIC6, we a in whole-cell currents The Cl− was and Cl− was with and with it was The of CLIC6 in and In we a current at as Cl− with a current C and was in the ions. that the of CLIC6 is Cl− CLICs are known to We also CLIC6 allow potassium ions. The was with and potassium a current was in HEK-293 with CLIC6 was in and to in CLIC6 was more In HEK-293 cells, we currents as recorded in the was with C and These that CLIC6 is Cl− channel. the regulation of channels by redox and pH has been H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar, K. et in a to chloride channels with to in 2002; PubMed Scopus Google Scholar, M. V. S. S. et of pH, and membrane protein in the regulation of in 2018; PubMed Scopus Google Scholar). pH is well characterized cellular membranes of pH chloride in Physiol. PubMed Scopus Google Scholar, J. F. S. H. pH regulation of and channels at the plasma membrane of PubMed Scopus Google Scholar). To the of pH CLIC6, we expressed CLIC6 in HEK-293 and whole-cell patch-clamp On pH from to we a but a change in current CLIC6 In addition to 10 μM IAA-94 C and we a of whole-cell currents in HEK-293 with CLIC6 at pH as in C and D. the was at a pH and in HEK-293 with a S. M. M. of in the of chloride intracellular channel 2012; PubMed Scopus Google that histidine in the C terminus are involved in conformational Two histidine in are to be involved in the conformational of their S. M. M. of in the of chloride intracellular channel 2012; PubMed Scopus Google Scholar). In CLIC6, histidine at 648 is with histidine We that is directly involved in a conformational change in We the to residue to role in the of CLIC6 by CLIC6 was in HEK-293 cells, and currents were recorded at pH and pH CLIC6 a in the current but the pH and was We also that CLIC6 was by IAA-94 and that is a residue involved in the conformational change of The of proteins is regulated by redox and is known to the channel function H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar, H. and redox regulation of the channel of using PubMed Scopus Google Scholar, S. et intracellular chloride channel protein a PubMed Scopus Google Scholar). We also the of In addition to in the the current at and In a the of the protein the membrane Rao S. Ponnalagu D. S. Singh H. S. et and of a of chloride Intracellular (CLIC) PubMed Scopus Google Scholar, H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar, H. and transmembrane PubMed Scopus Google Scholar, H. of intracellular and regulation by J. PubMed Scopus Google but we that the of the channel is by as the was of cysteine residue at in are the of redox regulation of H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar). we compared the and of cysteine the transmembrane of and CLIC6 In CLIC6, cysteine at is a residue of of H. regulation of by cysteine associated with the channel J. PubMed Scopus Google that this residue be a redox of to the of CLIC6 as reported H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar). In was to the CLIC6 we change in the of CLIC6 and The addition of 10 μM in the the of CLIC6, C and These residue in the regulation of CLIC6 CLIC6 was to be expressed in the and other of the it localizes with M. M. S. D. M. et of a novel member of the CLIC6, to PubMed Scopus Google Scholar, F. F. J. CLIC6, a member of the intracellular chloride channel with PubMed Scopus Google Scholar). We the of CLIC6 in from the of of CLIC6 in the mouse brain, and was by qRT-PCR, and was as The that was expressed in the in the brain, and in the and To determine are IAA-94 currents present in lung cells, we recorded currents from cells. of current was by IAA-94 and that are Cl− currents present in lung epithelial M. C. M. et of chloride currents in lung epithelial to Cell Physiol. PubMed Scopus Google Scholar). We also CLIC6 and and to CLIC6 from cells, and the CLIC6 after was by and Cl− currents were recorded from as they in and CLIC6 a Cl− current that was to The Cl− current a but by IAA-94 and In lung epithelial cells, other chloride channels and chloride channel were to be M. C. M. et of chloride currents in lung epithelial to Cell Physiol. PubMed Scopus Google but they as and are to IAA-94 M. C. M. et of chloride currents in lung epithelial to Cell Physiol. PubMed Scopus Google Scholar). These that CLIC6 is present and in cells. was discovered in in Molecular and of a novel chloride intracellular expressed in PubMed Scopus Google Scholar, D. of a in the cell associated with PubMed Scopus Google Scholar). was as a protein with a of and as D. of a in the cell associated with PubMed Scopus Google Scholar). The was found to be present in in associated with transport including the and the and was found to be present in the with a of the protein to the membrane in change in the of D. of a in the cell associated with PubMed Scopus Google Scholar). The to and it was the family as was CLIC6 and found to be in that are involved in transepithelial as and CLIC6 was to and it was from other CLICs in of N terminus and a 10 M. M. S. D. M. et of a novel member of the CLIC6, to PubMed Scopus Google Scholar). to other family CLIC6 is also present in the dimorphic soluble and membrane forms (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar). of Cl− from the to the membrane in cells, a in membrane translocation Molecular and of a novel chloride intracellular expressed in PubMed Scopus Google Scholar). On of Cl− was found to be as compared to be to the of a CLIC6 in Molecular and of a novel chloride intracellular expressed in PubMed Scopus Google Scholar). were the of CLIC6 as a of a Cl− channel. These were by the to currents in transepithelial of with CLIC6 M. M. S. D. M. et of a novel member of the CLIC6, to PubMed Scopus Google Scholar). In the it was that CLIC6 localizes to the and but the plasma membrane in M. M. S. D. M. et of a novel member of the CLIC6, to PubMed Scopus Google the of currents in cells. We this by CLIC6 in HEK-293 in the current and their in the plasma The and it of the and of proteins A. D. and of in with Cell PubMed Google Scholar). In channel of was discovered in the and Cl− channel was found in the cell membrane of F. M. and regulation of channels in PubMed Scopus Google Scholar). in cells, channel was F. M. and regulation of channels in PubMed Scopus Google the that the of CLIC6 in M. M. S. D. M. et of a novel member of the CLIC6, to PubMed Scopus Google be to the of of The CLIC6 is the protein all the and to other it has transmembrane To a CLIC6 to and the is to have a of H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar, and of the chloride intracellular channel protein membrane PubMed Scopus Google Scholar). play a role in the of CLICs the H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar, H. and redox regulation of the channel of using PubMed Scopus Google and have that cations also their the We have discovered that CLIC6 forms a channel with a and a at The is to CLIC6 and is other family (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar). The Cl− and is in of Cl− with a the of the channel. The of to the is known to the of of through the channel and is also channels channels: and PubMed Scopus (38) Google Scholar). Cl− in also the as the intracellular Cl− the physiological We also IAA-94 the CLIC6 as IAA-94 is known to the of family (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar). IAA-94 the CLIC6 at the and the of the channel. The currents were by These that CLIC6 is at The transmembrane of and CLIC6 are but the biophysical properties of channels are The in channel in CLIC6 other CLICs from the N terminus of CLIC6 and of CLIC6 as as reported Molecular and of a novel chloride intracellular expressed in PubMed Scopus Google Scholar). In HEK-293 by CLIC6, the addition of in the that channel The of was the cysteine residue of the transmembrane was by that the cysteine residue is also the as reported H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar). Chloride channels are known their (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar, K. et and of a chloride channel of cell PubMed Scopus Google Scholar). We in in HEK-293 cells. In the of K+ the currents of CLIC6 were from the recorded be to the of K+ channels in HEK-293 cations that the H. A. et and the PubMed Google and the of anions to the in channels H.C. of in J. Sci. 2021; PubMed Scopus Google Scholar). In CLICs are known to channels H. regulation of by cysteine associated with the channel J. PubMed Scopus Google and the of a smaller allow them to pass through but at a also the currents were chloride by and ions. These with (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar, H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar, H. and transmembrane PubMed Scopus Google Scholar, H. of intracellular and regulation by J. PubMed Scopus Google other CLICs that CLICs are channels with a of all the CLIC6 is Cl− ions. all the in the transmembrane are the and Cl− from other of the proteins various protein of the cellular of Cl− channels is cellular pH has been implicated in conformational in pH and S. M. M. of in the of chloride intracellular channel 2012; PubMed Scopus Google Scholar, S. from the it as J. Physiol. Cell Physiol. 2002; PubMed Scopus Google Scholar, K. L. Li H. et regulates cell and by and PubMed Scopus Google Scholar, S. of of soluble chloride intracellular channel protein at PubMed Scopus Google Scholar). In whole-cell pH was at pH and the cell was with pH we a in the pH IAA-94 but at pH we is a at pH the channel is from pH is the of pH to but at the pH, were The is a of the transmembrane and is at The of IAA-94 and currents CLIC6 change in the of the channel we discovered a histidine residue (H648) is all the CLICs and the currents and of CLIC6 currents was to We that the residue is also a pH in other are of the of other Cl− channels in HEK-293 H. C. of currents in the cell PubMed Scopus Google Scholar, J. H. S. channels expressed in PubMed Scopus Google at pH and are to but CLIC6 a in the current In in addition to cations L. J. D. F. et of membrane and chloride Cell Sci. PubMed Scopus Google was implicated in membrane of proteins H. regulation of by cysteine associated with the channel J. PubMed Scopus Google Scholar, S. et intracellular chloride channel protein a PubMed Scopus Google Scholar, et of the chloride intracellular channel the J. PubMed Scopus Google Scholar). and in the of a that the of CLIC6 membranes A. M. M. of CLIC6 by and 2018; PubMed Scopus Google Scholar). In HEK-293 with CLIC6, we recorded currents in of In whole-cell configuration, is to the we in the In change in the In a addition of to in CLIC6 A. M. M. of CLIC6 by and 2018; PubMed Scopus Google Scholar). with CLIC6 the of of CLIC6 that and the of are vital the of CLIC6 A. M. M. of CLIC6 by and 2018; PubMed Scopus Google Scholar). the channel is These that has the channel is in the membrane S. M. Chloride intracellular channel and Lett. 2010; 584: PubMed Scopus Google it the we in currents in the of was to and we cysteine involved in redox The cysteine residue at to cysteine in is all the proteins including (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar). CLIC6 was with we a in current at to the currents CLIC6 These the that CLIC6 is a and the also that the current is through CLIC6 and associated Cl− channel. In cells, we recorded Cl− currents were present in with CLIC6 The channel were from HEK-293 a of other proteins a of that IAA-94 channels are present in cells. The role of Cl− channels is is a of a Cl− channel The chloride channel is regulated by the transmembrane expressed in PubMed Scopus Google we CLIC6 to be a of a Cl− channel in lung cells. In we have that CLIC6 forms a Cl− channel is regulated by and pH also the role of a N terminus cysteine residue in the of CLIC6 in the cellular CLIC6 from was as a by the cysteine at to were to CLIC6 The was to the The was with at and cells. 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