Protein Kinase C-dependent Enhancement of Activity of Rat Brain NCKX2 Heterologously Expressed in HEK293 Cells

Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by β-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCϵ (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, β-phorbol myristate acetate (PMA), but not 4α-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events. Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by β-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCϵ (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, β-phorbol myristate acetate (PMA), but not 4α-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events. The family of Na+/Ca2++K+ exchangers (NCKX) 5The abbreviations used are: NCKX, Na+/Ca2+K+ exchanger; HEK, human embryonic kidney; PDBu, β-phorbol dibutyrate; PMA, β-phorbol myristate acetate; PKC, protein kinase C; PIE, PDBu-induced enhancement of NCKX2; TM, transmembrane segment; WT, wild type; fNCKX2, FLAG epitope-tagged NCKX2; PP, protein phosphatase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis; DN, dominant negative; CaT, calcium transient; XIP, exchanger inhibitory peptide. 5The abbreviations used are: NCKX, Na+/Ca2+K+ exchanger; HEK, human embryonic kidney; PDBu, β-phorbol dibutyrate; PMA, β-phorbol myristate acetate; PKC, protein kinase C; PIE, PDBu-induced enhancement of NCKX2; TM, transmembrane segment; WT, wild type; fNCKX2, FLAG epitope-tagged NCKX2; PP, protein phosphatase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis; DN, dominant negative; CaT, calcium transient; XIP, exchanger inhibitory peptide. catalyzes electrogenic exchange of four Na+ for one Ca2+ and one K+ across the plasma membrane (1Cervetto L. Lagnado L. Perry R.J. Robinson D.W. McNaughton P.A. Nature. 1989; 337: 740-743Crossref PubMed Scopus (285) Google Scholar, 2Dong H. Lights P.E. French R.J. Lytton J. J. Biol. Chem. 2001; 276: 25919-25928Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 3Szerencsei R.T. Prinsen C.F.M. Schnetkamp P.P.M. Biochemistry. 2001; 40: 6009-6015Crossref PubMed Scopus (34) Google Scholar). Although NCKX expression was originally considered to be restricted to retinal photo-receptors, new members of the NCKX family were identified from the brain and other excitable tissues (4Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 5Kraev A. Quednau B.D. Leach S. Li X.F. Dong H. Winkfein R. Perizzolo M. Cai X. Yang R. Philipson K.D. Lytton J. J. Biol. Chem. 2001; 276: 23161-23172Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 6Li X.F. Kraev A. 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NCX1 has been shown to be regulated by Ca2+, Na+, H+, the phospholipid environment of plasma membrane (15Hilgemann D.W. Ball R. Science. 1996; 273: 956-959Crossref PubMed Scopus (557) Google Scholar), and the action of protein kinases (16Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Many proteins related to phosphorylation can be co-immunoprecipitated with NCX1, including protein kinases, phosphatases, and A-kinase anchoring proteins. It was suggested that NCX1 and these signaling molecules comprise a macromolecular complex (17Schulze D.H. Muqhal M. Lederer W.J. Ruknudin A.M. J. Biol. Chem. 2003; 278: 28849-28855Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 18Katanosaka Y. Iwata Y. Kobayashi Y. Sibasaki F. Wakabayashi S. Shigekawa M. J. Biol. 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Neurosci. 2005; 25: 6057-6065Crossref PubMed Scopus (104) Google Scholar). It is known that synaptic by a which an enhancement of Ca2+ of the that synaptic and an increase in the of the X. Schneggenburger R. Nature. 2005; PubMed Scopus Google Scholar). The effect of on the Ca2+ however, to be that Ca2+ plays a role in the of synaptic in of PKC of NCKX be to have on Ca2+ and in on synaptic In the present we have the of neuronal NCKX family members by found that PKC activation and NCKX2 expressed in HEK293 whereas the activity of NCKX3 and was not and of PKC phosphorylation we have shown that Thr-476 and Ser-504 are involved in PKC-mediated activation of and embryonic cells were in with and in at The cells were with rat brain NCKX2 and a with The cells were used for the were the of the with a of were from which were with a were with an The was to The membrane current was at by a at and on a The current were a with a of The of the current was by the in the were and NCKX2 current perfusion The was from the current response to a of from to in and are by were at we used a BAPTA, with in NCKX2 was were the cells were with a and and with is to Na+/Ca2++K+ exchange was induced by of a which was of and with to was the to the with of were from the rat brain NCKX2 (4Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Abstract Full Text Full Text PDF PubMed Scopus (103) Google in the or double were to or with were by to of expression Lee R. Biol. PubMed Scopus Google was used to that the dominant negative mutant of PKC with a expression were by of PKC with a dominant negative mutation at the and for and into with Lee R. Biol. PubMed Scopus Google for The expression of in HEK293 cells was by was a expression of was by of HEK293 cells on were with and in a perfusion on a The was on a of cells by from at and with a The perfusion used either or together with and The cells were for with or with the perfusion with the cells were for with either β-phorbol myristate acetate (PMA), or for NCKX2 activity were by of the rate of of the In HEK293 cells in were with of and with in of for at The cells were with or or for at The cells were on with of and in with of and were at The was to a and on for by at for of protein was at for and of protein from the was to with the with protein and to a new The was with of by for by of of protein for The were three in by at for The was by of and to for The and protein were on and to The 32P was by the with in a one at for was of The were in and with the X. Lytton J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), by of The were and required of to The were of and 32P incorporation was by the of the 32P by the of the Ca2+ at the of the of the were from to M.H. Korogod N. Schneggenburger R. Ho W.K. Lee S.H. J. Neurosci. 2005; 25: 6057-6065Crossref PubMed Scopus (104) Google Scholar), a and calcium at when with and with an of of of were in an Ca2+ were by a an and a which were by a a with The for Ca2+ in have been in M.H. Korogod N. Schneggenburger R. Ho W.K. Lee S.H. J. Neurosci. 2005; 25: 6057-6065Crossref PubMed Scopus (104) Google Scholar). were by with a the of the of the and the of the The and at with were at and were from from and from inhibitory was by other including PDBu, alkaline and were from for the of NCKX2 in the of were to of from the The were compared are of cells Na+/Ca2++K+ in HEK293 NCKX2 current was at a of from HEK293 which were with a of H. Lights P.E. French R.J. Lytton J. J. Biol. Chem. 2001; 276: 25919-25928Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The Na+ Ca2+ in the of Ca2+ and To prevent the increase in which was to a of exchange in the The and which to be for cells current at when they were to Ca2+ and K+ but not with either was not significantly by that the current not HEK293 cells were they current To the cells were to from to the perfusion of or The from the the of which was distinct from the that the current to the electrogenic of Na+ in exchange for Ca2+ and K+ by the NCKX2 PDBu in a was by of with Li enhancement of was in In the of PDBu, however, was significantly and enhanced The enhancement the of The of in the and in the of PDBu that PDBu enhanced the current the to and was the that PDBu not current The for the in the of at of PDBu is shown in The of the PDBu effect on was the of at a the of PDBu, to the of or three the from the is to of PDBu enhanced NCKX2 activity in a in of at and PDBu of and of In the PDBu not NCKX2 activity suggesting that the effect of PDBu is on the activation of the of PDBu on NCKX from and cells The enhancement of NCKX2 activity by PDBu was significantly than that of NCKX3 or activity was little by The of was and for and of that the PDBu-induced enhancement (PIE) is for and a for an that in members of the NCKX family may regulation. The of NCKX2 by PDBu on the of that the effect of PDBu on was to activation of PKC, we PKC inhibitors prevent activation. PDBu was the enhanced to the not In when a PKC was to PDBu, the enhancement of was abolished for for Moreover, in the of the PKC of PDBu enhanced The of PKC inhibitors are in Although PDBu enhanced to of the the PIE was significantly inhibited by a PKC or by an for and PKC D. H. T. M. F. L. D. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.S. J. PubMed Scopus Google Scholar). To the of PKC in PIE, we the of PDBu in HEK293 cells that one of dominant negative of PKC together with The of was significantly reduced in the HEK293 cells the dominant negative for at of PDBu are in The of PIE was not significantly in the cells dominant negative or Consistently, of an of PKC and protein kinase had effect on PIE These suggest that PIE is by It has been reported that PKC can protein kinase and protein kinase T. M. R.J. Nature. PubMed Scopus Google Scholar, Y. J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, R.T. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). To investigate or protein kinase of PKC is involved in PIE, we or can of these enhanced suggesting that protein kinase is involved in PIE Moreover, a protein kinase had effect on PIE, suggesting that protein kinase is not involved in PIE of PDBu on Ca2+ at the of to PDBu can the NCKX activity expressed at the calyx of Held, a in the of the where NCKX, and the plasma membrane for and of the calcium M.H. Korogod N. Schneggenburger R. Ho W.K. Lee S.H. J. Neurosci. 2005; 25: 6057-6065Crossref PubMed Scopus (104) Google Scholar). calyx of K+ it is not to NCKX we the of PDBu on the rate at the calyx of was of PDBu and the rate of calcium by a To prevent a by of for we a on the calyx of a with a and the the we to of by axon a stimulation at the of the brain or action at were required for a was in the of To the activity of and plasma membrane we exchanger inhibitory and in the which and plasma membrane M.H. Korogod N. Schneggenburger R. Ho W.K. Lee S.H. J. Neurosci. 2005; 25: 6057-6065Crossref PubMed Scopus (104) Google Scholar, A. D.W. Philipson K.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Ca2+ is on the of from the we compared when the in the the is for Ca2+ and PDBu were and Ca2+ these were with a the of the at with the Ca2+ a that the activity of Ca2+ at the 11Kim M.H. Korogod N. Schneggenburger R. Ho W.K. Lee S.H. J. Neurosci. 2005; 25: 6057-6065Crossref PubMed Scopus (104) Google for The in with and was which was significantly than the and In the of and in the PDBu accelerated the rate of the Ca2+ transient and the to The calcium rate was from the of the decay of the Ca2+ transient and calcium of Ca2+ and and a of that the in the calcium decay rate Ca2+ and PDBu is at the NCKX activity is enhanced by of NCKX2 in HEK293 that PKC is a of the rat brain we PIE of a phosphorylation HEK293 cells were with alkaline phosphatase PDBu induced enhancement of and enhancement of the in the of alkaline phosphatase that the PDBu effect a phosphorylation of the NCKX2 protein was by of HEK293 cells by to The of that the incorporation of 32P into and the of expressed was and was by PKC activation. The shown in the of that NCKX2 from cells a of whereas was in from cells with PMA, but not with the 4α-PMA, in an of NCKX2 phosphorylation whereas the of NCKX2 present in the was The of had little effect on the enhanced phosphorylation. The from four a to 3-fold stimulation of 32P incorporation when to NCKX2 These suggest that the of NCKX2 phosphorylation was enhanced activation of PKC, whereas the of effect of that a of PKC was To that was in NCKX2 activity these Ca2+ of NCKX2 was by of HEK293 cells on with the and in a perfusion on a is shown in and from three are shown in NCKX2 activity was induced by a perfusion from to which an increase in Ca2+ and a in the cells not not that the rate of of the the to NCKX2 activity with perfusion and these a with induced an increase of in the rate of NCKX2 not with the These that in in NCKX2 activity was PDBu increase in 32P incorporation in response to stimulation was for three NCKX2 double PIE of not to the of the phosphorylation and the in were not to in the of phosphorylation the wild-type and mutant NCKX2 It was the of phosphorylation in these was than that of the wild Thr-476 in of the rat NCKX2 acid for PKC T. J. PubMed Scopus Google three in To which of these sites be by PDBu we putative PKC phosphorylation or to alanine, either individually or in combination. These were into the fNCKX2, and the PDBu response was in HEK293 cells To that these not expression and of we the NCKX current from cells wild-type or mutant in the current and that at the putative PKC phosphorylation either individually or in had on the and expression of we the effect of with PDBu on the of the of PDBu on the of together with current from wild-type and three of the were significantly from wild in of the of at of PDBu for for for for Although the of the mutant was enhanced by PDBu, PIE PDBu and the of to the of The response in the mutant that phosphorylation or other than Thr-476 by protein the of PDBu to the phosphorylation. To we the effect of inhibitors on the in PIE of in the mutant inhibitors were PDBu when the of are of and and the and to the found that acid a of and had effect on the of the other the inhibitors of or the in the of PDBu The of of PDBu PDBu acid PDBu or PDBu were or the in the PDBu and PDBu the wild-type and other single or in of with inhibitors It is known that activation of is on an increase in H. X. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google it was that was in the that the cells were with To to in we and by the to the used in the Indeed, was by a Ca2+ transient than Moreover, when was induced of the in PIE was not a These that the increase in induced by was to which in the but not the other the in PIE by that mutation at the three putative PKC phosphorylation sites completely inhibited the stimulation of induced by that multiple PKC consensus sites be involved in the stimulation of NCKX2 M. J. 1998; PubMed Scopus Google Scholar, H. L. W.G. J. PubMed Scopus Google Scholar). To were with three double and current for mutant and of with PDBu for and are shown in The double involving Thr-476 significantly reduced PIE of The of of PDBu were for for and for the other the PIE of the T166A/S504A mutant was inhibited to the that activation of be for the of PKC on the double we of on PIE of from the cells double was PDBu effect on was for of the double The of in the of PDBu were for for for and for T166A/S504A of effect that activation of was not related to the of PIE in the double The suggest that multiple PKC consensus sites are involved in PIE of To the of PKC to PIE, the for of of wild-type and at PDBu in the of were and on the in The PIE was in the T166A/S504A is the for mutant than in the is or is suggesting that these sites are not but to PIE with a In from the that PIE was in the mutant of and Ser-504 are than or or Ser-504 is it be that either or Ser-504 is not to PIE, but sites and to PIE. In the present we have the effect of PDBu on neuronal NCKX2 expressed in HEK293 and shown for the stimulation of NCKX2 activity by PKC, which may be via phosphorylation of the Recently, β-phorbol have been shown to not PKC but which of to the plasma membrane A. M. J. N. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). it that the PIE of NCKX2 we have is by to the plasma membrane by J.S. A. S. F. D. M. N. 2002; Full Text Full Text PDF PubMed Scopus Google for the PIE was not in other NCKX or be PIE were via a PIE of the NCKX2 current was by PKC inhibitors or or by of alkaline phosphatase in the NCKX2 double at PKC phosphorylation (T166A/T476A and T476A/S504A) abolished the PIE, suggesting a activation of NCKX2 by and enhanced phosphorylation of the exchanger protein was in the of it that NCKX family members expressed in the brain are regulated by To is the report on the of an NCKX family via protein kinases, are on in the PKC in PIE and are three of PKC to the of activation on calcium and In the present or three of were induced to the of in the of to the cells were with The to the of the enhanced in the of PDBu was suggesting that be involved in the PIE. with enhanced the 32P incorporation into NCKX2 of Moreover, we that of the dominant negative mutant of PKCϵ significantly reduced PIE. it was reported that a PKC but not PKC is expressed at the calyx of Held, where NCKX activity was M.H. Korogod N. Schneggenburger R. Ho W.K. Lee S.H. J. Neurosci. 2005; 25: 6057-6065Crossref PubMed Scopus (104) Google Scholar, N. T. T. S. A. 2001; Scopus (47) Google Scholar). The present that PIE may involve multiple PKC sites on the on of channels that PKC activity not by the of the but by the of of by PKC phosphorylation of the protein S. M. H. Biol. PubMed Scopus (28) Google Scholar, J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). the other of PKC of channels or of K+ phosphorylation of involved in signaling phosphorylation of the protein Neurosci. 2001; PubMed Scopus Google Scholar, 2003; Full Text Full Text PDF PubMed Scopus (103) Google Scholar). of and expression of phosphorylation of the D.H. H. G. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, J. X. J. Neurosci. PubMed Google Scholar). Although the of the PIE by mutation the in of the NCKX2 the mechanism that from phosphorylation of NCKX2 to enhanced be required to of new to the plasma membrane or the enhancement in activity of of NCKX2 we phosphorylation to of with the three double increase in 32P incorporation in response to was for double that PIE of not it was the of phosphorylation in these was significantly than that of the wild may for the the and phosphorylation of the NCKX2 NCKX2 may be in a at sites in to Thr-166, Thr-476, and Although these other sites not activity the incorporation of phosphorylation to activity The phosphorylation may at PKC sites that not the consensus or may be to PKC activation of a kinase with it is that the we have not phosphorylation have the effect of phosphorylation from the increase in activity via a in protein phosphorylation of Thr-476 is not a of PIE, suggest that Thr-476 plays a Moreover, the that PIE, the mutant was to that Thr-476 is not for the of PIE but for of PIE by other sites present on NCKX2 from by The PKC consensus involving Thr-166, Thr-476 or Ser-504 found in NCKX2 are not in NCKX3 or NCKX4. three of these NCKX family members putative sites in of the proteins that are to be The that NCKX2 was by PDBu that a of PKC consensus sites are in the proteins or that in exchanger from phosphorylation of In the present that multiple acid are involved in PKC-mediated stimulation of with Thr-476 in the the for enhancement of exchanger In the activity of NCKX3 and was not regulated by a involving family which is found in other an mechanism by which the of protein can be with

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