Permeation of Monovalent Cations through the Non-capacitative Arachidonate-regulated Ca2+ Channels in HEK293 Cells

In a manner similar to voltage-gated Ca2+ channels and Ca2+ release-activated Ca2+ (CRAC) channels, the recently identified arachidonate-regulated Ca2+ (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca2+ (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca2+ current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg2+. Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca2+ channels and store-operated Ca2+ channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances. In a manner similar to voltage-gated Ca2+ channels and Ca2+ release-activated Ca2+ (CRAC) channels, the recently identified arachidonate-regulated Ca2+ (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca2+ (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca2+ current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg2+. Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca2+ channels and store-operated Ca2+ channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances. store-operated Ca2+ Ca2+release-activated Ca2+ arachidonate-regulated Ca2+ arachidonic acid picofarad N-methyl-d-glucamine current/voltage Receptor-stimulated increases in Ca2+ entry in non-excitable cells are known to play a pivotal role in the generation and maintenance of the intracellular Ca2+ signals responsible for the control of such diverse functions as secretion, motility, growth, proliferation, and gene expression. However, despite extensive study, the mechanisms underlying such receptor-stimulated Ca2+ entry are currently far from clear. To date, most studies have focused on the so-called capacitative or store-operated model in which activation of Ca2+ entry occurs as a result of the emptying of intracellular Ca2+ stores (1Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2109) Google Scholar, 2Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1261) Google Scholar, 3Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1049) Google Scholar). Such entry is described as occurring via store-operated Ca2+(SOC)1 channels (4Parekh A.B. Penner R. Annu. Rev. Physiol. 1997; 77: 901-930Crossref Scopus (1291) Google Scholar, 5Lewis R.S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 279-307Crossref PubMed Scopus (83) Google Scholar). The ionic conductances associated with this store-operated entry were first characterized using patch-clamp techniques in mast cells and Jurkat lymphocytes (6Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1491) Google Scholar, 7Hoth M. Penner R. J. Physiol. ( Lond. ). 1993; 465: 359-386Crossref PubMed Scopus (661) Google Scholar, 8Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6259-6299Crossref Scopus (695) Google Scholar), where the resulting whole-cell currents were identified as I CRAC (for calciumrelease-activated calcium currents). These CRAC channels therefore represent the archetypal capacitative or store-operated channel. Although store-operated Ca2+ entry appears to be an almost ubiquitous feature of cells, the biophysical characterization of SOC channels from other cell types is rather limited, and the mechanism of activation of the channels is still unknown. Moreover, it seems unlikely that such channels are the exclusive route for the receptor-stimulated entry of Ca2+ in non-excitable cells (9Shuttleworth T.J. Cell Calcium. 1999; 25: 237-246Crossref PubMed Scopus (94) Google Scholar). Recently, we have identified a novel receptor-activated Ca2+ entry pathway that appears to be specifically responsible for the Ca2+entry associated with agonist stimulation at physiologically relevant concentrations (10Shuttleworth T.J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Full PubMed Scopus Google Scholar, T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar, T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar). pathway is of and is the generation of arachidonic acid non-capacitative Ca2+ entry have identified in a of cell including S. Cell Calcium. 1997; PubMed Scopus Google Scholar), cells Jr., J.W. J. Biol. Chem. 1999; Full Full PubMed Scopus Google Scholar), and cells J. and J. characterization of the conductance associated with this pathway in HEK293 cells stably transfected with the m3 muscarinic receptor that it certain that it from the store-operated current in the same cells T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar). ARC and CRAC channels, as as the endogenous SOC channels of cells the properties of with magnitude currents at and (4Parekh A.B. Penner R. Annu. Rev. Physiol. 1997; 77: 901-930Crossref Scopus (1291) Google Scholar, 5Lewis R.S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 279-307Crossref PubMed Scopus (83) Google Scholar, 7Hoth M. Penner R. J. Physiol. ( Lond. ). 1993; 465: 359-386Crossref PubMed Scopus (661) Google Scholar, T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar). The maintenance of the for Ca2+ these channels as voltage-gated Ca2+ is Ca2+ in the extracellular this for Ca2+ a feature of CRAC channels and voltage-gated Ca2+ channels is that external divalent cation concentrations to the in the of large monovalent currents through these channels M. Penner R. J. Physiol. ( Lond. ). 1993; 465: 359-386Crossref PubMed Scopus (661) Google Scholar, A. J. 1996; 7: Full Scopus Google J. Scholar). from voltage-gated Ca2+ channels that the for Ca2+ monovalent seen result from a of as from a rather from the of Ca2+ to the channel. In other the Ca2+ of these channels is similar in an of the monovalent of CRAC channels A. J. 1996; 7: Full Scopus Google Scholar, J. Scholar). we a similar in monovalent ion in the nominal absence of extracellular divalent in studies on the ARC channels T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar). the despite the diverse mechanisms of these channels arachidonic to similar mechanisms for In this study, we this feature in with the of to a for the ARC channels to the of them from the store-operated channels in the same cells. from the cell HEK293 that stably transfected with the m3 muscarinic receptor were a from of The cells were in with and in a at as T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar). were on that the of a patch-clamp at using the whole-cell A. J. Physiol. PubMed Scopus Google were at using an patch-clamp were from and to a of with internal currents were using from a of to were using from to were at to from a were at the and at the and at upon cell activation of I I were and for of current in the external were of through the patch-clamp of the and currents were compared to of the capacitative currents to a for the capacitative of To from these currents currents a were at a after the of the currents were at a The and with The Ca2+ of this to be as with R. in Scholar). or for the or internal the with or as The extracellular and and were from this for the divalent external and the with of the Ca2+ in this divalent external were using acid and compared The from to the with or as In external Ca2+ and of arachidonic acid to the in the activation of a current at with T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar), this current displayed a marked a and an absence of inactivation and of external with effects on the a to these these are with the characterization of this current as a current nominal removal of external divalent a current at seen to in the and in the this current to a of or the current seen in divalent external The current on the of as currents were seen to upon nominal removal of external divalent of In the absence of extracellular divalent the current in magnitude in In the divalent with divalent after the current in a of the current to with seen in We from to to the current/voltage of the current seen in the nominal absence of divalent cations. in the a of with and currents current at The a with marked at and at The nature of the that the current a current removal of extracellular divalent ions. the that of extracellular with the the of in the with in the complete absence of the magnitude of the current Moreover, currents were of extracellular and extracellular of these were the that the current a and that the large current seen upon nominal removal of extracellular divalent the of an monovalent of the of the current and the corresponding current a with currents through the same and currents were these divalent it is clear that the channels responsible are to and conductance to of ARC and SOC conductances for monovalent cations. ARC currents were activated of arachidonic acid and SOC currents were using a are the effects of with compared with the currents seen with as the cation in the and as the cation in the The effects of of in the external with or are for the monovalent currents through ARC and SOC the effects of of in the internal with or are for monovalent currents through ARC and SOC the effects of external on ARC and SOC currents at The external cation or the effects of internal on ARC and SOC currents at The internal cation or be seen in the corresponding and current the internal cation voltage-gated Ca2+ channels and the CRAC of non-excitable cells have to display a large to monovalent upon removal of extracellular divalent M. Penner R. J. Physiol. ( Lond. ). 1993; 465: 359-386Crossref PubMed Scopus (661) Google Scholar, A. J. 1996; 7: Full Scopus Google Scholar, J. Scholar). The of the monovalent current described are with the of a similar in the ARC channels we have To we compared the magnitude of the monovalent current at with that of the corresponding current in the same cell to removal of extracellular divalent cations. The a between the of the two currents the that the of a of channels ARC the of this to the of the non-capacitative arachidonate-regulated conductance that it from store-operated or capacitative conductances. The of the monovalent conductance that upon of external divalent in the store-operated conductance of Jurkat lymphocytes have characterized A. J. 1996; 7: Full Scopus Google Scholar, J. Scholar). However, it appears that store-operated channels represent a of conductances of CRAC is it to be to the conductance with the endogenous SOC conductance in the same cell We therefore the of removal of divalent from the extracellular in cells in which the internal Ca2+ stores with a In the Ca2+ and a current at upon whole-cell this current displayed the with characterization as a store-operated current including marked and T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar). currents were in cells in which intracellular were in for T.J. J. Biol. Chem. Full Full PubMed Scopus Google or of the in the to divalent external an in the current at to a of or the current in divalent external store-operated current in the absence of extracellular divalent a in magnitude in a of for the store-operated current in the nominal absence of divalent were of from to in the and in the these a current that at with and currents current at of at and at were also apparent, the a The of the and currents in cells were Moreover, the store-operated currents were extracellular and and currents were upon to divalent in the of extracellular with the currents, the of in the with in the complete absence of the magnitude of the current on these we conclude that the currents are the result of a and that the currents seen upon nominal removal of extracellular divalent after of the intracellular represent the of the endogenous store-operated conductance of the magnitude of the SOC current in seen in the of divalent in the with that of the corresponding current seen in the same cell upon removal of external divalent ions. that the of the two currents were to other the it is clear that the endogenous store-operated or capacitative conductance and the non-capacitative conductance display a marked to monovalent upon nominal removal of extracellular divalent a property with voltage-gated Ca2+ channels and the store-operated Ca2+ conductance The most between the currents displayed ARC and SOC channels is the monovalent conductance I ARC SOC the whole-cell currents for the two conductances in are similar in magnitude at the monovalent ARC currents seen upon removal of extracellular divalent are the corresponding monovalent SOC currents at Such a a of ARC and SOC channels to the in divalent external However, such have to be that for CRAC channels for Ca2+ of currents S. Cell Calcium. 1997; PubMed Scopus Google Scholar). Moreover, as the monovalent SOC currents displayed a spontaneous the decline in the monovalent ARC current and the decline in the monovalent SOC current appears to of of it is that the inactivation the SOC channels is occurring with the activation of the monovalent upon removal of extracellular divalent and could therefore to the monovalent current seen with these these it is clear the store-operated CRAC channels M. Penner R. J. Physiol. ( Lond. ). 1993; 465: 359-386Crossref PubMed Scopus (661) Google Scholar, A. J. 1996; 7: Full Scopus Google Scholar, J. Scholar), the endogenous SOC channels and the ARC channels of HEK293 cells are of monovalent Ca2+ be in this between the ARC and SOC SOC and I ARC in the nominal absence of extracellular divalent displayed with and monovalent currents the in the and in the Moreover, the in the with I SOC ARC displayed These that SOC and ARC channels have a to and these To the monovalent cation of the ARC and SOC channels in were monovalent cation substitutions in the and The are and and in of the of monovalent conductance for the two channels were from the resulting current of in the with a on the current of the ARC channels at and external were the current at to and currents were also substitutions of the external of external with the current at to with the current to the of the external ion the of the internal ion to current through the ARC We have the for these effects in are of an of the the with the a conductance pathway in Scholar). on these of current a for the monovalent conductance of the ARC channels of is are from in However, in the the of the currents and the that the were rather the such of the in a similar of in the current at upon of external with seen with the SOC current and However, as the magnitude of the currents be the rate of the inactivation In this the rate of the spontaneous inactivation of the SOC current in the of external magnitude to in a of the SOC current in the of external at in from the rate of inactivation seen these of external with in an of the current to a similar to that seen with ARC currents In marked to the of ARC currents, currents through SOC channels were of external with or The a monovalent for currents in the channels of despite the in of the in an of were also to the monovalent in the internal of internal the current at through the ARC channels to in current at seen with SOC channels internal to and this could be an on the rate of spontaneous as this in the to inactivation of internal with in the complete absence of currents through ARC or SOC channels and on these for current a monovalent cation for ARC and SOC channels of is despite the of the in an of The of the currents and for ARC and channels is seen in the CRAC channels of Jurkat cells the same A. J. 1996; 7: Full Scopus Google Scholar, J. Scholar). However, it that internal the magnitude of the currents the of the Jurkat CRAC channels such that the currents are J. Scholar). also that large currents in the absence of internal that are in the of internal J. Scholar). We therefore the effects of removal of internal on the monovalent currents for ARC and the of in the and in the removal of internal on the magnitude of the currents through ARC channels in the magnitude of the current seen the external cation However, of the that removal of internal the of in the current internal also the current at from to and monovalent SOC currents were internal on currents, these were or the ARC currents, removal of internal the of of the current Moreover, in marked to the seen in ARC currents, removal of internal the current at from to and and J. that removal of internal the spontaneous inactivation of the monovalent current through the CRAC channels in Jurkat cells. However, such in the endogenous SOC channels of HEK293 cells the in the and in the In these cells, the to a decline in the current at were in the of internal and in absence the monovalent current a rather spontaneous decline in magnitude after activation upon removal of extracellular divalent such of the cells to divalent with that the current also However, of the cells to external divalent a of the current a to divalent that this of the current also associated with a of the monovalent The that the of these two currents and were to other this that the two currents the of the same of in the CRAC channels of Jurkat cells A. J. 1996; 7: Full Scopus Google Scholar). mechanism for this spontaneous decline and in with the similar decline for monovalent CRAC currents A. J. 1996; 7: Full Scopus Google Scholar, J. Scholar), we have described this as a of of such a is with the to the current to extracellular divalent ions. The rate of decline in the corresponding monovalent ARC current after activation seen with the currents In after the decline in the monovalent ARC current the cells to external divalent for to a of the monovalent ARC current to of the monovalent These that the decline in the monovalent ARC current a rather from that seen with To between these two rather and with the to ARC currents upon to extracellular divalent we to this decline in the monovalent ARC current as a rather an the of this is to the of the current on to divalent in the for and of the monovalent current seen of the cell to divalent external However, of to a cell in which the monovalent SOC current in the of a large monovalent current The of this current to the activation of SOC current resulting from to the internal Ca2+ as an monovalent current could be activated after spontaneous inactivation of SOC currents that in or of in the The magnitude of the current in cells stores or from that of the monovalent ARC current and the magnitude of the monovalent SOC current in the same cell Moreover, it displayed the spontaneous decline described for the monovalent ARC These that the ARC currents be activated in a cell SOC currents have and that the two currents therefore represent the of distinct conductances. 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These monovalent currents are and extracellular Ca2+ and are the same that the Ca2+ Moreover, in the magnitude of the monovalent current is to the magnitude of the corresponding Ca2+ these that the Ca2+ currents in the of extracellular divalent and the monovalent currents in the nominal absence of divalent the of the same The Ca2+ current and large monovalent current of CRAC channels is therefore a feature and ARC certain differences are in the of this between these We have the of the channels and in ARC and channels have similar current compared with the CRAC channels of Jurkat cells for ARC and channels and for CRAC A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6259-6299Crossref Scopus (695) Google Scholar, T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar), ARC channels have monovalent current Jurkat CRAC current for CRAC channels from to Jr., J.W. J. Biol. Chem. 1999; Full Full PubMed Scopus Google Scholar). 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Although this seen in the currents, of the other of the inactivation of monovalent CRAC currents were seen in the currents, including the to the monovalent currents after a of to external Ca2+ currents in divalent and the monovalent currents in divalent which in as for CRAC channels A. J. 1996; 7: Full Scopus Google Scholar). rather of in the monovalent currents through the ARC the inactivation seen in the currents, and after to to external to a of the monovalent ARC Although we have on the for this such is of a of the currents rather an characterization of these two and the mechanisms we were to the of to extracellular Ca2+ for the of the currents after spontaneous inactivation to that ARC currents could be activated of extracellular in cells in which the monovalent SOC currents The activated monovalent currents displayed the properties of ARC ARC channels be activated in a cell SOC channels have with that the two conductances are and distinct T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar, T.J. J. Biol. Chem. 1999; Full Full PubMed Scopus Google Scholar). the that are and marked differences in the of the CRAC channels of Jurkat cells, the endogenous SOC channels of HEK293 cells, and non-capacitative ARC We have described the and differences between the and ARC channels in T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar). In this study, we have as these channels the property of to a monovalent upon removal of extracellular divalent cations. first the two store-operated conductances CRAC and it is clear are similar in several also to display marked of these differences that the effects of internal on the two of the most differences between the CRAC and conductances are current and monovalent However, these differences CRAC currents are in the absence of internal J. Scholar). In other channels CRAC channels in the absence of internal that the between these two store-operated conductances in the or absence of or in for Mg2+. this the of differences in the of or differences in the of the characterization at the However, it seems that these two store-operated conductances are of is to be a of similar to the endogenous SOC channels and ARC channels in the same cells a between these two conductances. several marked differences are These differences in the magnitude of the of monovalent to Ca2+ currents, in the rate and nature of the spontaneous decline in monovalent and in the effects of external monovalent cation substitutions and of internal removal on these that ARC and SOC currents the of distinct conductances. Moreover, we have that monovalent ARC currents be activated after the complete spontaneous inactivation of SOC currents in the same cell. this on the and nature of the currents for these two conductances T.J. J. Biol. Chem. Full Full PubMed Scopus Google and on distinct to T.J. J. Biol. Chem. 1999; Full Full PubMed Scopus Google Scholar). the most and between these two conductances is SOC channels, ARC channels are activated T.J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar). the displayed these two conductances in monovalent described with the currents are that in the of the of the channels in the of at the as for CRAC channels 1999; PubMed Scopus Google Scholar, Jr., J.W. J. Biol. Chem. Full Full PubMed Scopus Google Scholar). We for and and for and and on an of the