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

Abstract

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...

Regulation of Cardiac L-Type Ca 2+ Channel Ca V 1.2 Via the β-Adrenergic-cAMP-Protein Kinase A Pathway

“…However, smaller or no effects on basal I Ca were seen in later works that used cell dialysis of inhibitors through the patch pipette or extracellular incubation with cell-permeable inhibitors. The results are divergent, from no effect of Rp-cAMPS (0.1-2 mmol/L in the pipette) and H-89 in adult mouse cardiomyocytes, 92,104,105 through a minor ≈20% reduction in basal I Ca by dialysis of purified PKI protein (2.9 µmol/L) in adult guinea pig myocytes, 88 to 20% to 55% inhibition in late versus early embryonic mouse cardiomyocytes, respectively, after extracellular application of 15 µmol/L of a cell-permeable PKI peptide. 72 Interestingly, reduction in basal I Ca caused by PKA inhibitors 72 or protein phosphatase PP2A 106 was correlated with a decrease in phosphorylation of Ser1928, monitored with a site-specific antibody.…”

“…Unlike the effect of Iso, the enhancement of basal I Ca by phosphatase inhibitors is not blocked by PKA inhibitors. 92 ; however, a PKC inhibitory peptide was without effect. 92 In all, basal I Ca in mammalian cardiomyocytes is regulated by a balanced action of serine/threonine kinases and phosphatases 104,106,113 ; the kinases involved remain to be identified.…”

“…92 ; however, a PKC inhibitory peptide was without effect. 92 In all, basal I Ca in mammalian cardiomyocytes is regulated by a balanced action of serine/threonine kinases and phosphatases 104,106,113 ; the kinases involved remain to be identified. Notably, phosphorylation and dephosphorylation also regulate the level of the membrane phosphatidyl inositol biphosphate, a ubiquitous regulator of ion channels and transporters.…”

“…Direct intracellular perfusion (dialysis) of the myocytes through the patch pipette often yielded incomplete inhibition of the β-AR effect: 50% to 70% with 1 µmol/L PKI, 0.5 mmol/L Rp-cAMPS, or a cocktail of PKA and phosphorylation inhibitors. [87][88][89][90] Other works reported full inhibition of effects of β-AR agonists in frog and rat ventricular cardiomyocytes by dialyzing cells with 2 to 5 mmol/L Rp-cAMPS, 16 µmol/L PKI (which is >3 orders of magnitude higher than its K i ), 20 µmol/L PKI peptide (PKI [15][16][17][18][19][20][21][22] ; K i =36 nmol/L), [91][92][93] or with 1 µmol/L H-89 applied externally. 94 Although the latter studies used rather high doses of inhibitors, the accumulated data strongly support the view that a major part and possibly all of the β-AR enhancement of cardiac Ca V 1.2 is mediated by activation of PKA.…”

Mechanisms of L-Type Ca 2+ Current Downregulation in Rat Atrial Myocytes During Heart Failure

“…29 Mechanisms other than PDE, such as the enhanced phosphatase activity, can also contribute to the low basal cAMPdependent regulation of I Ca . 30 Indeed, as in other species and myocyte types, [31][32][33] the I Ca of rat atrial myocytes is highly sensitive to phosphatase inhibition; these enzymes appear to play an important role in the basal regulation of the current, keeping with the tight coupling between Ca 2ϩ channels and OA-sensitive phosphatases. 34 Various studies have shown that the kinetics of I Ca inactivation also depend on cAMPdependent regulation of the current 35,36 and the slowly decaying I Ca in HF may be another consequence of its downregulation.…”