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• W1965034333 abstract "To test the hypothesis that ATP activation of BK channels in GH3 cells involves cytosolic phospholipase A2 (cPLA2) as a potential protein target for phosphorylation, we first inhibited the activity of cPLA2 by both pharmacologic and molecular biologic approaches. Both approaches resulted in a decrease rather than an increase in BK channel activity by ATP, suggesting that in the absence of cPLA2, phosphorylation of other regulatory elements, possibly the BK channel protein itself, results in inactivation rather than activation of the channel. The absence of changes in activity in the presence of the non-substrate ATP analog 5′-adenylyl-β,γ-imidodiphosphate verified that ATP hydrolysis was required for channel activation by ATP. Experiments with an activator and inhibitor of protein kinase C (PKC) support the hypothesis that PKC can be involved in the activation of BK channels by ATP; and in the absence of PKC, other kinases appear to phosphorylate additional elements in the regulatory pathway that reduce channel activity. Our data point to cPLA2-α (but not cPLA2-γ) as one target protein for phosphorylation that is intimately associated with the BK channel protein. To test the hypothesis that ATP activation of BK channels in GH3 cells involves cytosolic phospholipase A2 (cPLA2) as a potential protein target for phosphorylation, we first inhibited the activity of cPLA2 by both pharmacologic and molecular biologic approaches. Both approaches resulted in a decrease rather than an increase in BK channel activity by ATP, suggesting that in the absence of cPLA2, phosphorylation of other regulatory elements, possibly the BK channel protein itself, results in inactivation rather than activation of the channel. The absence of changes in activity in the presence of the non-substrate ATP analog 5′-adenylyl-β,γ-imidodiphosphate verified that ATP hydrolysis was required for channel activation by ATP. Experiments with an activator and inhibitor of protein kinase C (PKC) support the hypothesis that PKC can be involved in the activation of BK channels by ATP; and in the absence of PKC, other kinases appear to phosphorylate additional elements in the regulatory pathway that reduce channel activity. Our data point to cPLA2-α (but not cPLA2-γ) as one target protein for phosphorylation that is intimately associated with the BK channel protein. protein kinase C cytosolic phospholipase A2 adenosine 5′-O-(thiotriphosphate) 5′-adenylyl-β,γ-imidodiphosphate polymerase chain reaction phorbol 12-myristate 13-acetate Long-term modulation of ion channels is beginning to be appreciated. This concept suggests that changes in ion channel properties are not dependent on continued occupation of a receptor by an agonist, but rather arise via some long-lasting metabolic modification such as protein phosphorylation (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 2Levitan I.B. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 3-22Crossref PubMed Google Scholar, 3Armstrong D.L. Rossie S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: ix-xxCrossref PubMed Google Scholar). For this process to occur, the receptor and ion channel do not have to be intimately associated, but communicate some signal transduction pathway activated by occupancy of the receptor. Such a signal transduction pathway, which may involve several steps, ultimately results in a modification that alters the activity and persists until this modification is reversed (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar). Protein phosphorylation and dephosphorylation have been shown to be important in the modulation of a number of ion channels, particularly those in the central nervous system (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 2Levitan I.B. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 3-22Crossref PubMed Google Scholar, 3Armstrong D.L. Rossie S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: ix-xxCrossref PubMed Google Scholar, 4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar). One important type of central nervous system ion channel that has been shown to be modulated by phosphorylation/dephosphorylation is the large conductance Ca2+-activated potassium (BK) channel, with at least six functionally distinct types having been described within the central nervous system (5Reinhart P.H. Chung S. Levitan I.B. Neuron. 1989; 2: 1031-1041Abstract Full Text PDF PubMed Scopus (184) Google Scholar). BK channels are important to such widely diverse central nervous system functions as neural regulation of the heart originating in the nucleus tractus solitarius and sleep, which is dependent on repetitive rhythmic activity originating in the reticular formation of the thalamus (6Dekin M.S. Getting P.A. J. Neurophysiol. ( Bethesda ). 1987; 58: 215-229Crossref PubMed Scopus (79) Google Scholar, 7Golomb D. Wang K.J. Rinzel J. J. Neurophysiol. ( Bethesda ). 1994; 72: 1109-1126Crossref PubMed Scopus (148) Google Scholar, 8McCormick D.A. Pape H.C. J. Physiol. ( Lond. ). 1990; 431: 291-318Crossref PubMed Scopus (844) Google Scholar). BK channels are also involved in neuropeptide secretion, regulation of presynaptic calcium signals, and neurotransmitter release (9Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar). Because of the physiologic importance of BK channels, we have been particularly interested in the cellular signaling mechanisms responsible for controlling the activity of BK channels (10Denson D.D. Worrell R.T. Eaton D.C. Am. J. Physiol. Cell Physiol. 1996; 270: C636-C644Crossref PubMed Google Scholar, 11Denson D.D. Worrell R.T. Middleton P. Eaton D.C. Am. J. Physiol. 1999; 276: C201-C209Crossref PubMed Google Scholar).It is well documented that BK channels that have been reconstituted in lipid bilayers can be either activated or inhibited by the addition of ATP, protein kinases, or protein phosphatases (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar, 9Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar, 12Campbell W.B. Gebremedhin D. Pratt P.F. Harder D.R. Circ. Res. 1996; 78: 415-423Crossref PubMed Scopus (1101) Google Scholar, 13Reinhart P.H. Chung S. Martin B.L. Brautigan D.L. Levitan I.B. J. Neurosci. 1991; 11: 1627-1635Crossref PubMed Google Scholar). In fact, BK channels have been classified as either type I or II based on their response to the catalytic subunit of the cAMP-dependent protein kinase (4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar). Type II BK channels in mammalian brain reconstituted in lipid bilayers are activated by ATP and ATP analogs via an endogenous protein kinase activity intimately associated with the channel. For these channels, it appears the kinase involved is protein kinase C (PKC)1 since activators of PKC enhance the response to ATP, whereas inhibitors of PKC reverse the response to ATP (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar). BK channels in GH3 cells appear to belong to the class that is activated in the presence of ATP with or without the addition of exogenous protein kinase, whereas BK channels in GH4 cells appear to be inactivated by ATP or protein kinases and activated by protein phosphatases (14White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Crossref PubMed Scopus (228) Google Scholar, 15Shipston M.J. Armstrong D.L. J. Physiol. ( Lond. ). 1996; 493: 665-672Crossref PubMed Scopus (63) Google Scholar). Investigators examining BK channels that are activated in response to ATP in lipid bilayers concluded that there must be an endogenous protein kinase activity intimately associated with these channels (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar, 9Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar, 13Reinhart P.H. Chung S. Martin B.L. Brautigan D.L. Levitan I.B. J. Neurosci. 1991; 11: 1627-1635Crossref PubMed Google Scholar). However, because BK channels in lipid bilayers are at infinite dilution, the phosphorylation target could be either the channel protein itself or a regulatory protein that is intimately associated with the ion channel. The possibility that the action of the kinase may be on a regulatory protein rather than (or in addition to) the channel protein itself is intriguing. One important regulatory protein for BK channels in GH3 cells is cytosolic phospholipase A2 (cPLA2) (10Denson D.D. Worrell R.T. Eaton D.C. Am. J. Physiol. Cell Physiol. 1996; 270: C636-C644Crossref PubMed Google Scholar, 11Denson D.D. Worrell R.T. Middleton P. Eaton D.C. Am. J. Physiol. 1999; 276: C201-C209Crossref PubMed Google Scholar). Until recently, it has been thought that optimal activation of cPLA2 requires both Ca2+i and phosphorylation (16Qiu Z.H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar, 17Qiu Z.H. Gijon M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 18Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 19Gijón M.A. Leslie C.C. J. Leukocyte Biol. 1999; 65: 330-336Crossref PubMed Scopus (249) Google Scholar). It was also thought that cPLA2 resided in the cytosol and translocated to the cell membrane only in response to large increases in [Ca2+]i. It is now clear that the mechanisms involved in the regulation of cPLA2, including the relative importance of phosphorylation, [Ca2+]i, and the site of subcellular localization, show considerable variability in different cell types and even between different agonists in the same cell type. However, in most cells, there is a significant pool of cPLA2 constitutively associated with the membrane capable of producing arachidonic acid (17Qiu Z.H. Gijon M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 20Gijon M.A. Spencer D.M. Leslie C.C. Adv. Enzyme Regul. 2000; 40: 255-268Crossref PubMed Scopus (27) Google Scholar). In addition, recent evidence suggests that there is more than one isoform of cPLA2 (21Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.L. J. Biol. Chem. 1998; 273: 21926-21932Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) that responds to intracellular calcium and other stimuli differently.In this investigation, we sought to determine whether cPLA2could be a potential target for phosphorylation, resulting in subsequent activation of BK channels. To test this hypothesis, we studied the response of wild-type BK channels in GH3 cells to ATP, to the poorly hydrolyzable analog ATPγS, and to the non-hydrolyzable substrate AMP-PNP. These results were compared with responses from BK channels that had been exposed to either aristolochic acid or antisense oligonucleotides to cPLA2 prior to the addition of ATP. Since PKC has been reported to be involved in the activation of reconstituted BK channels as well as the phosphorylation of cPLA2, we examined the possibility that PKC could be associated with the ATP response of BK channels in GH3cells. Finally, since two isoforms of cPLA2,viz. cPLA2-α (86 kDa) and cPLA2-γ (60 kDa), have now been identified, we also sought to verify that the cPLA2-α isoform is the one closely associated with BK channels in GH3 cells (21Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.L. J. Biol. Chem. 1998; 273: 21926-21932Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar).DISCUSSIONThe major findings in this investigation are as follows. 1) ATP results in a significant activation of BK channels in wild-type GH3 cells. 2) Inhibition of cPLA2 by aristolochic acid blocks the activation of BK channels by ATP in wild-type GH3 cells. 3) Reducing the expression of cPLA2 with antisense oligonucleotides also suppresses the activation of BK channels by ATP. 4) Activation of PKC results in a significant potentiation of the activation in BK channels produced by ATP. 5) Inhibition of PKC in wild-type GH3 cells results in a significant decrease in BK channel activity when ATP is added. 6) Inhibition of cPLA2 blocks the activation of BK channels by PKC. 7) cPLA2-α appears to be the isoform associated with BK channels in GH3 cells.Protein phosphorylation and dephosphorylation are important in the modulation of central nervous system BK channels. (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 2Levitan I.B. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 3-22Crossref PubMed Google Scholar, 3Armstrong D.L. Rossie S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: ix-xxCrossref PubMed Google Scholar, 4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar). However, modulation of BK channels by phosphorylation is complicated. BK channels reconstituted in lipid bilayers can be either activated or inhibited by the addition of ATP, protein kinases, or protein phosphatases (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar, 12Campbell W.B. Gebremedhin D. Pratt P.F. Harder D.R. Circ. Res. 1996; 78: 415-423Crossref PubMed Scopus (1101) Google Scholar). In fact, BK channels have been classified as either type I or II based on their response to phosphorylation (4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar). Type II BK channels in mammalian brain reconstituted in lipid bilayers are activated by ATP and ATP analogs via an endogenous protein kinase activity intimately associated with the channel. For these reconstituted channels, it appears that the kinase involved is similar to protein kinase C since activators of PKC enhance the response to ATP, whereas inhibitors of PKC reverse the response to ATP (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar, 9Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar, 13Reinhart P.H. Chung S. Martin B.L. Brautigan D.L. Levitan I.B. J. Neurosci. 1991; 11: 1627-1635Crossref PubMed Google Scholar). Besides a PKC-like protein, Levitan and co-workers (27Schopperle W.M. Holmqvist M.H. Zhou Y. Wang J. Wang Z. Griffith L.C. Keselman I. Kusinitz F. Dagan D. Levitan I.B. Neuron. 1998; 20: 565-573Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 28Esguerra M. Wang J. Foster C.D. Adelman J.P. North R.A. Levitan I.B. Nature. 1994; 369: 563-565Crossref PubMed Scopus (97) Google Scholar, 29Wang J. Zhou Y. Wen H. Levitan I.B. J. Neurosci. (online). 1999; 19 (:1–7): RC4Crossref PubMed Google Scholar) have shown a close association between BK channels and a several other regulatory proteins that directly alter BK activity. BK channels in GH3 cells appear to belong to the class that is activated in the presence of ATP with or without the addition of exogenous protein kinase, whereas BK channels in GH4 cells (a subclone of GH3 cells) appear to be inactivated by ATP or protein kinases and activated by protein phosphatases (9Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar, 14White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Crossref PubMed Scopus (228) Google Scholar, 15Shipston M.J. Armstrong D.L. J. Physiol. ( Lond. ). 1996; 493: 665-672Crossref PubMed Scopus (63) Google Scholar). To test this hypothesis, we studied the responses of wild-type BK channels to ATP in GH3 cells.BK Channels in GH3 Cells Are Activated by MgATP and ATPγS, but Not by AMP-PNPMgATP and ATPγS caused theP o of BK channels in wild-type GH3 cells to increase significantly. The non-substrate ATP analog AMP-PNP did not result in any significant change in P o, further suggesting that the effects produced by ATP require ATP hydrolysis for channel activation (30Ashcroft F.M. Annu. Rev. Neurosci. 1988; 11: 97-118Crossref PubMed Scopus (767) Google Scholar). This is different from the ATP-sensitive ion channels found in a variety of tissues in which ATP functions as a ligand to alter channel properties by binding reversibly to an allosteric site, but without ATP hydrolysis (30Ashcroft F.M. Annu. Rev. Neurosci. 1988; 11: 97-118Crossref PubMed Scopus (767) Google Scholar). The magnitude of the increase noted for ATPγS was significantly larger than that noted for MgATP presumably because there was a phosphorylation/dephosphorylation equilibrium established in the MgATP experiments, whereas the reaction of ATPγS is only slowly reversible (9Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar, 13Reinhart P.H. Chung S. Martin B.L. Brautigan D.L. Levitan I.B. J. Neurosci. 1991; 11: 1627-1635Crossref PubMed Google Scholar). This activation could be due to a direct kinase-mediated phosphorylation of the channel by ATP. Alternatively, since PLA2 production of arachidonic acid is also a potent activator of BK channels in excised patches and since PLA2 can be activated by phosphorylation, ATP activation of BK channels could be due to kinase-mediated phosphorylation of PLA2 rather than direct phosphorylation of BK channels. Although these data clearly show that ATP hydrolysis and subsequent protein phosphorylation are required for activation of BK channels in GH3 cells, the target for phosphorylation is still unclear.Inhibition of cPLA2 Blocks the Activation of BK Channels by ATP in Wild-type GH3 CellsInvestigators examining BK channels that are activated in response to ATP in lipid bilayers concluded that there must be an endogenous protein kinase activity intimately associated with these channels (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar, 9Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar, 13Reinhart P.H. Chung S. Martin B.L. Brautigan D.L. Levitan I.B. J. Neurosci. 1991; 11: 1627-1635Crossref PubMed Google Scholar). However, the phosphorylation target could be either the channel protein itself or a regulatory protein that is intimately associated with the ion channel (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 2Levitan I.B. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 3-22Crossref PubMed Google Scholar, 4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar, 9Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar, 13Reinhart P.H. Chung S. Martin B.L. Brautigan D.L. Levitan I.B. J. Neurosci. 1991; 11: 1627-1635Crossref PubMed Google Scholar). We have shown that one important regulatory protein for BK channels in GH3 cells is cPLA2 (11Denson D.D. Worrell R.T. Middleton P. Eaton D.C. Am. J. Physiol. 1999; 276: C201-C209Crossref PubMed Google Scholar). Optimal activation of cPLA2requires both Ca2+i and phosphorylation (16Qiu Z.H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar, 17Qiu Z.H. Gijon M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 18Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 19Gijón M.A. Leslie C.C. J. Leukocyte Biol. 1999; 65: 330-336Crossref PubMed Scopus (249) Google Scholar). In this investigation, we sought to determine whether cPLA2could be a potential target for phosphorylation, resulting in subsequent activation of BK channels. To test this hypothesis, we used a pharmacologic and an antisense approach to lower cPLA2activity in wild-type GH3 cells. Aristolochic acid caused a significant reduction in P o for BK channels. This observation is consistent with our previous reports (11Denson D.D. Worrell R.T. Middleton P. Eaton D.C. Am. J. Physiol. 1999; 276: C201-C209Crossref PubMed Google Scholar). Subsequent addition of MgATP caused the P o to decrease, although this decrease was not statistically significant. The addition of aristolochic acid to patches in which BK channels were activated by ATP resulted in a significant decrease in activity. These data suggested that cPLA2 was one target for phosphorylation by ATP. Because aristolochic acid is a pharmacologic inhibitor of cPLA2 and could possibly be producing its effect on the MgATP response via some other mechanism, we examined the effects of MgATP on excised patches derived form cells treated with cPLA2 antisense oligonucleotides. As previously reported, treatment of wild-type GH3 cells in the present investigation with antisense oligonucleotides resulted in an ∼90% decrease in the expression of cPLA2 by both biochemical and Western blot analyses (11Denson D.D. Worrell R.T. Middleton P. Eaton D.C. Am. J. Physiol. 1999; 276: C201-C209Crossref PubMed Google Scholar). Exposure of excised patches from antisense oligonucleotide-treated cells resulted in a modest, albeit significant decrease in BK channel activity. These data support the hypothesis that ATP phosphorylation of cPLA2 is one important pathway for the activation of BK channels by ATP. Our data further suggest that, in the absence of cPLA2, there may be additional targets for phosphorylation that act to inhibit BK channel activity like the inhibition seen in GH4 cells (14White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Crossref PubMed Scopus (228) Google Scholar, 15Shipston M.J. Armstrong D.L. J. Physiol. ( Lond. ). 1996; 493: 665-672Crossref PubMed Scopus (63) Google Scholar).PKC Is Involved in the Activation of BK Channels by ATPType II BK channels in mammalian brain reconstituted in lipid bilayers are activated by ATP and ATP analogs via an endogenous protein kinase activity intimately associated with the channel. For these channels, it appears that the kinase involved is similar to protein kinase C since activators of PKC enhance the response to ATP, whereas inhibitors of PKC reverse the response to ATP (1Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Crossref PubMed Google Scholar, 4Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar, 9Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar, 13Reinhart P.H. Chung S. Martin B.L. Brautigan D.L. Levitan I.B. J. Neurosci. 1991; 11: 1627-1635Crossref PubMed Google Scholar). Since PKC could activate cPLA2 either directly or via an alternative kinase pathway, we studied the effect of MgATP on BK channels in the presence of the specific PKC inhibitor GF 109203X. Treatment of excised patches with GF 109203X had no effect on the P o of BK channels. However, the addition of MgATP to cells treated with GF 109293X resulted in a significant decrease in P o. This decrease was similar to that observed in cells treated with antisense oligonucleotides, where the addition of MgATP also decreased theP o. On the other hand, treatment of cells that had been exposed to MgATP with PMA, an activator of PKC, resulted in a significant increase in P o. On the other hand, for BK channels in cells treated with cPLA2 antisense oligonucleotides, exposure to PMA did not result in any significant change in P o. These results suggest that 1) PKC is necessary for ATP activation of BK channels; 2) in the absence of PKC, other kinases appear to phosphorylate additional elements in the regulatory pathway that reduce channel activity; 3) PLA2 is partially phosphorylated and active in GH3 cells under basal conditions; and 4) PLA2 is a target for PKC phosphorylation. (However, our data cannot exclude the possibility that mitogen-activated protein kinase also plays a role in the activation of cPLA2, as suggested by Leslie and co-workers (16Qiu Z.H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar, 31Gijon M.A. Spencer D.M. Siddiqi A.R. Bonventre J.V. Leslie C.C. J. Biol. Chem. 2000; 275: 20146-20156Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar).)cPLA2-α Rather than cPLA2-γ Appears to Be the Isoform Associated with BK Channels in GH3CellsRecently, a novel membrane-associated cPLA2isoform (cPLA2-γ) has been described (21Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.L. J. Biol. Chem. 1998; 273: 21926-21932Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) (in contrast to the original isoform, termed cPLA2-α). cPLA2-γ is associated with the plasma membrane, but it lacks the [Ca2+]i-dependent phospholipid-binding domain found in cPLA2-α, so its activation is entirely [Ca2+]i-independent. Although cPLA2-γ shares significant sequence homology with cPLA2-α, the absence of the phospholipid-binding domain reduces the size to ∼60 kDa, so cPLA2-γ can be easily distinguished on Western blots from the ”classical“ type IV cPLA2 (cPLA2-α). Several lines of evidence argue against cPLA2-γ being the isoform associated with BK channels in GH3 cells. First, a GenBankTM/EBI Data Bank search using the sequences for the sense and antisense oligonucleotides used in this study did not identify cPLA2-γ (or any other sequence besides cPLA2-α). Second, Western blots do not have a band at 60 kDa, where cPLA2-γ should run. Finally, PCR amplification of GH3 cell mRNA using primers that would amplify any cPLA2 isoforms in GH3 cells resulted in the amplification of only cPLA2-α. This is entirely consistent with the tissue distribution of cPLA2-γ, which showed very little of this isoform in brain or brain-derived tissues (21Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.L. J. Biol. Chem. 1998; 273: 21926-21932Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). These data support our claim that the results with cells treated with antisense oligonucleotides were, in fact, due solely to the depletion of cPLA2-α.A Portion of the cPLA2-α Pool in GH3Cells Resides in the Cell Membrane of Wild-type CellsUntil recently, it has been thought that cPLA2 resides almost exclusively in the cytosol and translocates to the cell membrane (and other membranes) only when stimulated by large increases in [Ca2+]i. If this were true, then it would be difficult to reconcile with our observation that cPLA2activity appears to be constitutively associated with the small patches of plasma membrane excised with our patch pipettes. An explanation of this apparent discrepancy involves the observation by Osterhout and Shuttleworth (26Osterhout J.L. Shuttleworth T.J. J. Biol. Chem. 2000; 275: 8248-8254Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) that at least a portion of the cellular pool of cPLA2-α resides in the cell membrane and appears to be activated without increases in [Ca2+]i. Our data showing that the relative amount of cPLA2-α is higher in GH3 cell membranes than in the cytosol are consistent with this previous observation. It should be noted that although the PLA2 assay employed here does not distinguish between secretory and cytosolic PLA2, the Western blot analysis and PLA2 amplification data described earlier suggest that cytosolic PLA2 is the major form of the enzyme present in GH3 cells. Some investigators have suggested that the membrane-associated cPLA2-α is stabilized in the membrane by interaction with certain anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (32Leslie C.C. Channon J.Y. Biochim. Biophys. Acta. 1990; 1045: 261-270Crossref PubMed Scopus (99) Google Scholar, 33Mosior M. Six D.A. Dennis E.A. J. Biol. Chem. 1998; 273: 2184-2191Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). We do not question the validity of the widely reported [Ca2+]i-dependent activation of some fraction of the type IV cPLA2 that is known to be associated with the [Ca2+]i-dependent translocation of the enzyme to the membrane. Our data suggest, however, that activation of PLA2 associated with BK channels may specifically involve a pool of cPLA2 that is already located at or near its substrate in the membrane and is bound to phosphatidylinositol 4,5-bisphosphate or other phospholipids.In summary, our data point to cPLA2-α as one target protein for phosphorylation that is intimately associated with the BK channel protein. Although we cannot unequivocally rule out the BK channel protein itself as an additional target for phosphorylation, the data that show a decrease in BK channel activity in the presence of ATP and the absence of cPLA2 suggest that direct phosphorylation of the channel protein would serve to decrease rather than increase activity. Long-term modulation of ion channels is beginning to be appreciated. This concept suggests tha" @default.
• W1965034333 created "2016-06-24" @default.
• W1965034333 creator A5005624414 @default.
• W1965034333 creator A5011685660 @default.
• W1965034333 creator A5028548827 @default.
• W1965034333 creator A5072040832 @default.
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• W1965034333 date "2001-03-01" @default.
• W1965034333 modified "2023-10-18" @default.
• W1965034333 title "Cytosolic Phospholipase A2 Is Required for Optimal ATP Activation of BK Channels in GH3 Cells" @default.
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