Regulation by phosphorylation of purified epithelial Na+ channels in planar lipid bilayers

Oh, Young S.; Smith, Peter; Bradford, A. L.; Keeton, Deborah A.; Benos, Dale J.

doi:10.1152/ajpcell.1993.265.1.c85

Cited by 51 publications

(29 citation statements)

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“…It thus seems likely that PKC exerts a tonic inhibitory effect on sodium channel activity as has been previously suggested (3,26). The finding that FK506 has no effect on sodium transport or PKC activity, although it binds tightly to FKBP12, represents another example of the peculiar effects determined by the immunophilin-immunophilin ligand complex.…”

Section: Table III Rap-fkbp12 Inhibits Pkc Activitymentioning

confidence: 65%

Rapamycin Inhibits Protein Kinase C Activity and Stimulates Na+ Transport in A6 Cells

Rokaw

West

Johnson

1996

Journal of Biological Chemistry

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Rapamycin and FK506 have unique cellular effects despite the fact that they bind to the same set of immunophilins, the FK506 binding proteins (FKBP). We have previously reported that rapamycin (RAP) stimulates sodium transport in A6 cells. FK506 did not stimulate sodium transport but did inhibit the stimulation seen in RAP-treated cells. Since FKBP12 has been shown to have sequence homology with an endogenous inhibitor of protein kinase C (PKC) and PKC inhibition has been shown to increase Na ؉ channel activity in A6 cells, studies to determine the effect of RAP on PKC activity and its relationship to sodium transport were performed. Here we report that RAP stimulates sodium transport, and the effect is not additive to that seen with a cellpermeant inhibitor of PKC␣ and -␤ subtypes. RAP significantly inhibits endogenous PKC activity in A6 cells both in membrane and cytosolic preparations. There is a strong correlation between the degree of inhibition of PKC activity and the stimulation of sodium transport by RAP. RAP has no effect on Na Tacrolimus (FK506), cyclosporin, and rapamycin (RAP) 1 are known to have effects on T-cell activation and proliferation. These effects are mediated through the interaction of the drug with its binding protein or immunophilin. The immunophilins are divided into two broad classes, the FK506 binding proteins (FKBPs) and the cyclophilins, both of which are characterized by peptidyl-prolyl cis-isomerase (rotamase) activity. The FK506 binding proteins bind both FK506 and RAP and are named for their molecular weights (FKBP12, FKBP56, FKBP25). The cyclophilins bind the structurally distinct macrocyclic peptide cyclosporin. The immunosuppressive effects of these agents are not mediated solely by the drug themselves or by the immunophilin to which they bind. Rather the binding of drug to the immunophilin exposes an effector region on the drug which then inhibits downstream pathways (1, 2). At present it is know that two distinct drugs that bind to the same immunophilin can have drastically different effects. For example, although FK506 and RAP both bind with high affinity to FKBP12 only FK506 inhibits calcineurin (1, 2). In contrast, the RAP⅐FKBP complex has been found to inhibit cell proliferation by inhibiting p70 S6 kinase activation (22). A6 cells, derived from Xenopus laevis are a well characterized, high resistance epithelia with transport characteristics similar to the mammalian cortical collecting duct (3). Na ϩ channel activity is known to be increased in response to aldosterone (4, 23), insulin (5), and vasopressin (4, 6 -9), each acting through unique pathways. In addition, inhibition of protein kinase C (PKC) activity has been shown to increase sodium channel activity in cell-attached patches of A6 (3). We have recently reported that RAP stimulates transepithelial sodium transport in A6 (10). The effect is seen within 15 min of addition and persists for 4 h. An excess of FK506 had no effect on basal transport but completely inhibited the RAP-induced effect. This suggested that ...

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Section: Table III Rap-fkbp12 Inhibits Pkc Activitymentioning

confidence: 65%

Rapamycin Inhibits Protein Kinase C Activity and Stimulates Na+ Transport in A6 Cells

Rokaw

West

Johnson

1996

Journal of Biological Chemistry

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“…Activation of PKC greatly diminished Na ϩ transport in A6 cells (52,53) and in the LLC-PK1 epithelial cell line (54). PKC also attenuated the activity of purified renal amiloride-sensitive sodium channels (55). Contrary to these outcomes, PKC activation had no effect on Na ϩ transport in the rat cortical collecting duct (56) and even stimulated sodium transport across frog skin epithelium (57).…”

Section: Rt-pcr and Western Blot Detection Of Pkc Isozymes-rt-mentioning

confidence: 99%

Protein Kinase C Isoform Antagonism Controls BNaC2 (ASIC1) Function

Berdiev¹,

Xia²,

Jovov³

et al. 2002

Journal of Biological Chemistry

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We explored the involvement of protein kinase C (PKC) and its isoforms in the regulation of BNaC2. Reverse transcriptase PCR evaluation of PKC isoform expression at the level of mRNA revealed the presence of ␣ and ⑀/⑀ in all glioma cell lines analyzed; most, but not all cell lines expressed ␦ and . No messages were found for the ␤I and ␤II isotypes of PKC in the tumor cells. Normal astrocytes expressed ␤ but not ␥. The essential features of these results were confirmed at the protein level by Western analysis. This disproportionate pattern of PKC isoform expression in glioma cell lines was further echoed in the functional effects of these PKC isoforms on BNaC2 activity in bilayers. PKC holoenzyme or the combination of PKC␤I and PKC␤II isoforms inhibited BNaC2. Neither PKC⑀ nor PKC or their combination had any effect on BNaC2 activity in bilayers. The inhibitory effect of the PKC␤I and PKC␤II mixture on BNaC2 activity was abolished by a 5-fold excess of a PKC⑀ and PKC combination. PKC holoenzymes, PKC␤I, PKC␤II, PKC␦, PKC⑀, and PKC phosphorylated BNaC2 in vitro. In patch clamp experiments, the combination of PKC␤I and PKC␤II inhibited the basally activated inward Na ؉ conductance. The variable expression of the PKC isotypes and their functional antagonism in regulating BNaC2 activity support the idea that the participation of multiple PKC isotypes contributes to the overall activity of BNaC2.The recent molecular identification of a class of proton-sensitive ion channels (ASIC; acid-sensitive ion channel (1, 2); also called BNC and BNaC; brain Na ϩ channel (3, 4)) belonging to the degenerin (DEG)/ENaC superfamily of ion channels (5) added a new molecular entity to the already complicated field of nociception. Even though their participation in nociception is controversial, ASICs might underlie some properties of native proton-induced currents (6 -8) and could contribute to the function of nociceptive transduction with many other key constituents including nociceptor-specific voltage-gated Na ϩ channels, ATP-gated channels, and capsaicin receptors (9 -13).The distinguishing feature of this family of ion channels, namely, proton-induced conductance, is exhibited by ASIC1a (14), BNaC2 (4), ASIC1b (15), ASIC␤ (16), ASIC2 (or BNC1) (3), BNaC1 (4), MDEG (17), and ASIC3 (DRASIC; 18), hASIC3 (19,20), and hTNaC1 (21). However, ASIC4 is functionally inactive (22, 23) and may require an association with an accessory protein(s) and/or other subunit(s) of the family, like the splice variant form of ASIC2 (ASIC2b, also called MDEG2) (24) or ASIC␤2, a splice variant of ASIC␤ (25). The multiplicity of current responses to extracellular acid loads in different neurons is consistent with the existence of functionally distinct ASICs in these cells.The tissue distribution of the ASIC members is not limited to the nervous system but also includes many other tissues such as the lung, testis, and intestine (20,21,26,27). Sensory neuron-specific expression of DRASIC has been reported (18), but Chen et al. (16) have found low level trans...

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“…Immunoprecipitation of iodinated cell surface proteins with an anti-antiamiloride antibody in A6 epithelia suggested that vasopressin increased antigen expression at the cell surface (25). However, no increase in surface expression was found using an antibody against a biochemically purified Na + channel (26). An important limitation of these studies is that the molecular identity of the protein(s) recognized by the antibodies and their relationship to ENaC are unknown.…”

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confidence: 99%

Liddle’s syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na+ channel to the cell surface

2000

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Regulation by phosphorylation of purified epithelial Na+ channels in planar lipid bilayers

Cited by 51 publications

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Rapamycin Inhibits Protein Kinase C Activity and Stimulates Na+ Transport in A6 Cells

Rapamycin Inhibits Protein Kinase C Activity and Stimulates Na+ Transport in A6 Cells

Protein Kinase C Isoform Antagonism Controls BNaC2 (ASIC1) Function

Liddle’s syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na+ channel to the cell surface

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