Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart.

Hwang, Tzyh Chang; Horie, Minoru; Gadsby, David C.

doi:10.1085/jgp.101.5.629

Cited by 107 publications

(75 citation statements)

References 43 publications

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“…6, A and B). These results confirm those of Hwang et al (7) and suggest that the difference between our result and theirs is due to higher phosphatase activity at 36°, which results in a partial dephosphorylation of I Cl .…”

Section: Resultssupporting

confidence: 92%

“…The regulation of this channel, like that of the Ca 2ϩ channel, involves two phosphorylation sites. One site is dephosphorylated by protein phosphatase 2A, and the other is dephosphorylated by a phosphatase with a low sensitivity to microcystin and okadaic acid, which may be protein phosphatase 2C (7). In addition, the channel is regulated by ATP binding and hydrolysis by its two nucleotide binding domains.…”

Section: Discussionmentioning

confidence: 99%

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Effects of Protein Phosphatase and Kinase Inhibitors on Ca²⁺and Cl⁻Currents in Guinea Pig Ventricular Myocytes

Hirayama

Hartzell

1997

Mol Pharmacol

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SUMMARYIt is well-established that in heart, both the L-type Ca 2ϩ channel and the cystic fibrosis transmembrane conductance regulator Cl Ϫ channel are regulated by cAMP-dependent phosphorylation. However, it is not clear whether both of these channels are regulated in concert by protein kinase A (PKA) or whether there are mechanisms that independently control the phosphorylation of these two PKA targets. The purpose of this study was to compare the effects of various protein phosphatase and protein kinase inhibitors on these two ionic currents (I Ca and I Cl ) in guinea pig ventricular myocytes to gain insight into these questions. We found that both the stimulation and washout of the effects of isoproterenol on I Cl are about twice as fast as the effects on I Ca , probably because the dephosphorylation reaction for I Cl is faster than that for I Ca . In contrast, inhibition of protein phosphatases with 10 M microcystin stimulated both I Ca and I Cl , but the stimulation of I Cl was much slower and smaller than the stimulation of I Ca . The effect of microcystin was inhibited by staurosporine (K i ϭ 171.5 and 161 nM for I Ca and I Cl , respectively), suggesting that the stimulation was due to a kinase. The kinase was not protein kinase C (PKC) because it was not inhibited by the specific pseudosubstrate inhibitor of PKC, PKC (19 -31) , and it was not PKA because it was not inhibited by adenosine 3Ј,5Ј-cyclic phosphorothioate. These results suggest that although both the Ca 2ϩ and Cl Ϫ channels are regulated by cAMP-dependent phosphorylation, another protein kinase may also regulate these channels, and the kinetics of the response of the channels to phosphorylation can be modulated independently by protein phosphatases.In mammalian cardiac myocytes, ␤-adrenergic agonists regulate a variety of ionic currents, including I Ca and I Cl (1-3). Both currents are stimulated by cAMP-dependent phosphorylation via the G s /adenylyl cyclase/cAMP/PKA cascade, but both of these currents may be regulated by phosphorylation of more than a single phosphorylation site.In the case of I Ca , Tsien et al. (4) proposed that two different phosphorylation sites were responsible for the ␤-adrenergic regulation of Ca 2ϩ channel availability (N) and channel gating (P o ). Support for this hypothesis has come from studies on rabbit ventricular myocytes in which different concentrations of okadaic acid selectively affect N and p o (5) and from our studies on frog ventricular myocytes in which the amplitude of I Ca is regulated by two phosphorylation sites that can be distinguished by the phosphatases that dephosphorylate them (6). One site is dephosphorylated by phosphatase 2A, and the other site is dephosphorylated by a phosphatase with a low sensitivity to the phosphatase inhibitors microcystin, okadaic acid, and calyculin A.The regulation of I Cl is even more complicated (3). The regulation of this channel, like that of the Ca 2ϩ channel, involves two phosphorylation sites. One site is dephosphorylated by protein phosphatase 2A, and t...

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Section: Resultssupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Effects of Protein Phosphatase and Kinase Inhibitors on Ca²⁺and Cl⁻Currents in Guinea Pig Ventricular Myocytes

Hirayama

Hartzell

1997

Mol Pharmacol

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“…The procedures for isolation and storage of Guinea pig ventricular myocytes were described previously (Hwang et al, 1993). All patch-clamp experiments were done using an AxoPatch 200B, a Digidata 1200 A/D board, and pClamp (6 or 8) software (all from Molecular Devices, Sunnyvale, CA).…”

Section: Cftr Experimentsmentioning

confidence: 99%

“…Whole-cell recordings were done at ϳ36°C, using wide-tipped (0.8-1.5 M⍀) borosilicate glass (PG52151-4; WPI, Sarasota, FL) pipettes with an intrapipette perfusion device inserted as described previously (Hwang et al, 1993). The pipette solution for intracellular dialysis contained 85 mM aspartic acid, 5 mM pyruvic acid, 10 mM EGTA, 20 mM tetraethylammonium-Cl, 2 mM MgCl 2 , 5 mM Tris 2 -creatine phosphate, 10 mM MgATP, 0.1 mM Tris 2.5 -GTP, 10 mM HEPES, 5.5 mM glucose (buffered to pH 7.4 with CsOH).…”

Section: Cftr Experimentsmentioning

confidence: 99%

2,3-Butanedione Monoxime Affects Cystic Fibrosis Transmembrane Conductance Regulator Channel Function through Phosphorylation-Dependent and Phosphorylation-Independent Mechanisms: The Role of Bilayer Material Properties

Artigas¹,

Al’Aref²,

Hobart³

et al. 2006

Mol Pharmacol

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2,3-Butanedione monoxime (BDM) is widely believed to act as a chemical phosphatase. We therefore examined the effects of BDM on the cystic fibrosis transmembrane regulator (CFTR) Cl Ϫ channel, which is regulated by phosphorylation in a complex manner. In guinea pig ventricular myocytes, forskolinactivated whole-cell CFTR currents responded biphasically to external 20 mM BDM: a rapid ϳ2-fold current activation was followed by a slower ( ϳ20 s) inhibition (to ϳ20% of control). The inhibitory response was abolished by intracellular dialysis with the phosphatase inhibitor microcystin, suggesting involvement of endogenous phosphatases. The BDM-induced activation was studied further in Xenopus laevis oocytes expressing human epithelial CFTR. The concentration for half-maximal BDM activation (K 0.5 ) was state-dependent, ϳ2 mM for highly and ϳ20 mM for partially phosphorylated channels, suggesting a modulated receptor mechanism. Because BDM modulates many different membrane proteins with similar K 0.5 values, we tested whether BDM could alter protein function by altering lipid bilayer properties rather than by direct BDM-protein interactions. Using gramicidin channels of different lengths (different channel-bilayer hydrophobic mismatch) as reporters of bilayer stiffness, we found that BDM increases channel appearance rates and lifetimes (reduces bilayer stiffness). At 20 mM BDM, the appearance rates increase ϳ4-fold (for the longer, 15 residues/monomer, channels) to ϳ10-fold (for the shorter, 13 residues/monomer channels); the lifetimes increase ϳ50% independently of channel length. BDM thus reduces the energetic cost of bilayer deformation, an effect that may underlie the effects of BDM on CFTR and other membrane proteins; the state-dependent changes in K 0.5 are consistent with such a bilayer-mediated mechanism.

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“…Dephosphorylation is also involved in the regulation of CFTR, and at least some of the phosphatases controlling the process appear to be localized to the plasma membrane (14). For instance, protein phosphatase 2A (PP2A) affects CFTR activity to a greater extent than protein phosphatases 1 or 2B (PP1, PP2B) in National Institutes of Health 3T3 fibroblasts stably expressing recombinant CFTR (20), whereas CFTR expressed in cardiac myocytes appears to be controlled by PP2A and PP2C (21). These examples illustrate that CFTR regulation is dependent on specific isoforms of several proteins associated with the cAMP-PKA pathway.…”

Section: Introductionmentioning

confidence: 99%

Activation of endogenous deltaF508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition.

et al. 1996

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Many heterologously expressed mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) exhibit residual chloride channel activity that can be stimulated by agonists of the adenylate cyclase/protein kinase A pathway. Because of clinical implications for cystic fibrosis of activating mutants in vivo, we are investigating whether ⌬ F508, the most common disease-associated CFTR mutation, can be activated in airway epithelial cells.

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Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart.

Cited by 107 publications

References 43 publications

Effects of Protein Phosphatase and Kinase Inhibitors on Ca²⁺and Cl⁻Currents in Guinea Pig Ventricular Myocytes

Effects of Protein Phosphatase and Kinase Inhibitors on Ca²⁺and Cl⁻Currents in Guinea Pig Ventricular Myocytes

2,3-Butanedione Monoxime Affects Cystic Fibrosis Transmembrane Conductance Regulator Channel Function through Phosphorylation-Dependent and Phosphorylation-Independent Mechanisms: The Role of Bilayer Material Properties

Activation of endogenous deltaF508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition.

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Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart.

Cited by 107 publications

References 43 publications

Effects of Protein Phosphatase and Kinase Inhibitors on Ca2+and Cl−Currents in Guinea Pig Ventricular Myocytes

Effects of Protein Phosphatase and Kinase Inhibitors on Ca2+and Cl−Currents in Guinea Pig Ventricular Myocytes

2,3-Butanedione Monoxime Affects Cystic Fibrosis Transmembrane Conductance Regulator Channel Function through Phosphorylation-Dependent and Phosphorylation-Independent Mechanisms: The Role of Bilayer Material Properties

Activation of endogenous deltaF508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition.

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Effects of Protein Phosphatase and Kinase Inhibitors on Ca²⁺and Cl⁻Currents in Guinea Pig Ventricular Myocytes

Effects of Protein Phosphatase and Kinase Inhibitors on Ca²⁺and Cl⁻Currents in Guinea Pig Ventricular Myocytes