PKA mediates constitutive activation of CFTR in human sweat duct

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels are constitutively activated in sweat ducts. Since phosphorylation-dependent and -independent mechanisms can activate CFTR, we sought to determine the actual mechanism responsible for constitutive activation of these channels in vivo. We show that the constitutively activated CFTR Cl(-) conductance (gCFTR) in the apical membrane is completely deactivated following alpha-toxin permeabilization of the basolateral membrane. We investigated whether such inhibition of gCFTR following permeabilization is due to the loss of cytoplasmic glutamate or due to dephosphorylation of CFTR by an endogenous phosphatase in the absence of kinase activity (due to the loss of kinase agonist cAMP, cGMP or GTP through alpha-toxin pores). In order to distinguish between these two possibilities, we examined the effect of inhibiting the endogenous phosphatase activity with okadaic acid (10(-8) M) on the permeabilization-induced deactivation of gCFTR. We show that okadaic acid (1) inhibits an endogenous phosphatase responsible for dephosphorylating cAMP but not cGMP or G protein-activated CFTR and (2) prevents deactivation of CFTR following permeabilization of the basolateral membrane. These results indicate that distinctly different phosphatases may be responsible for dephosphorylating different kinase-specific sites on CFTR. We conclude that the phosphorylation by PKA alone appears to be primarily responsible for constitutive activation of gCFTR in vivo.

Fig. 1

Effect of α-toxin and cAMP on transepithelial electrical properties. a A representative electrical trace showing the effect of permeabilization of basolateral membrane with α-toxin. In this experiment, lumen and bath were perfused with 150 mM NaCl and 140 mM KGlu + 5 mM ATP, respectively. The amiloride-sensitive ENaCs in the apical membrane were blocked by amiloride. Under these conditions, the lumen positive transepithelial potential and conductance predominantly reflect Cl⁻ conductance. Notice that α-toxin permeabilization caused progressive loss of transepithelial electrical conductance and lumen positive electrical potential. Hence, the experimental changes in the electrical properties induced by α-toxin permeabilization of the basolateral membrane primarily reflect loss of CFTR Cl⁻ conductance, which is the predominant Cl⁻ channel in this native tissue, as previously mentioned. The α-toxin-induced deactivation of transepithelial Cl⁻ conductance is reactivated by the addition of the CFTR agonist cAMP in the presence of a physiological concentration of ATP. b Summary of similar experiments as shown in a. Notice that α-toxin-induced deactivation of Cl⁻ conductance is fully restored following application of cAMP + ATP in the cytoplasmic bath. (C) and (L) represent cytosolic bath and lumen, respectively. The data reflect mean ± SE. * Significantly different from values before application of α-toxin and after application of α-toxin + cAMP: P > 0.001

Fig. 2

Lack of effect of α-toxin on the electrical properties of sweat duct in the absence of Cl⁻. a A representative electrical trace showing effect of α-toxin in the complete absence of Cl⁻ in the lumen and bath. In this experiment α-toxin was applied after replacing the luminal Cl⁻ with impermeant anion gluconate. Notice that replacing luminal Cl⁻ with gluconate has a qualitatively similar effect as that induced by α-toxin (i.e., loss of transepithelial electrical conductance and lumen positive electrical potential). These results further indicated that α-toxin permeabilization inhibited apical CFTR Cl⁻ conductance. Activation of CFTR Cl⁻ conductance was indicated by the changes in the amplitude of transepithelial current pulse indicated by downward voltage deflections and the lumen positive transepithelial Cl⁻ diffusion potential induced by 150 mM Cl⁻ in the lumen and 140 mM impermeant anion gluconate (Glu) in the bath. b Summary of the results obtained from similar experiments as shown in a. Notice that transepithelial potential and conductance were significantly reduced in the absence of Cl⁻, which did not decrease any further following application of α-toxin. (C) and (L) represent cytosolic bath and lumen, respectively. * Significantly different from corresponding values in the presence of luminal Cl⁻ before application of α-toxin: P > 0.001

Fig. 3

a Effect of α-toxin on the transepithelial potential and conductance in ΔF508 homozygous CF ducts. In this experiment the lumen was perfused with 150 mM NaCl + amiloride and the bath was perifused with 140 mM KGlu + 5 mM ATP before α-toxin application. Notice that there is a significantly smaller depolarization of transepithelial potential, reflecting absence of CFTR in the plasma membranes of this tissue. In contrast to the non-CF ducts, application of α-toxin had little effect either on the transepithelial potential or on the conductance. b In contrast to the homozygous CF ducts, heterozygous CF ducts with R117H/ΔF508 mutations showed a small but consistent depolarization to the imposed Cl⁻ gradient. In addition, application of α-toxin resulted in a qualitatively similar response (abolition of transepithelial potential and conductance) as in normal ducts, but the magnitude of response was significantly smaller. These results are consistent with the fact that R117H CF mutation retains residual Cl⁻ conductance. (C) and (L) represent cytosolic bath and lumen, respectively

Fig. 4

Effect of okadaic acid on α-toxin-induced deactivation of gCFTR. a A representative electrical trace showing the effect of PP2A inhibitor okadaic acid on the α-toxin-induced deactivation of gCFTR. This experiment was designed to test the hypothesis that deactivation of CFTR Cl⁻ conductance following α-toxin permeabilization of the basolateral membrane is due to uninterrupted phosphatase dephosphorylation of CFTR in the absence of PKA phosphorylation activity (due to loss of cAMP through α-toxin pores). Hence, inhibiting the endogenous phosphatase responsible for dephosphorylating CFTR should prevent deactivation of CFTR Cl⁻ conductance following α-toxin permeabilization. Notice that, consistent with this hypothesis, prior inhibition of phosphatase (most likely PP2A) with okadaic acid prevented CFTR deactivation after application of α-toxin. CFTR remained activated as long as ATP was present in the cytoplasmic bath. b Summary of the data from similar experiments as shown in a. (C) and (L) represent cytosolic bath and lumen, respectively. The data indicate mean ± SE. Notice that after treating the duct with okadaic acid, α-toxin failed to deactivate CFTR-Cl⁻ conductance

Fig. 5

Effect of glutamate on α-toxin-induced deactivation of CFTR. a A representative electrical trace showing the effect of glutamate on α-toxin-induced deactivation of gCFTR. Notice that the transepithelial conductance and Cl⁻ diffusion potentials remained relatively unaffected by α-toxin permeabilization in the presence of 1 mM α-ketoglutarate in the cytosol. b In comparison to the lack of effect of α-toxin as shown in a, α-toxin permeabilization spontaneously decreased transepithelial Cl⁻ conductance and diffusion potential in the absence of glutamate or its metabolite, which could be reversed immediately following application of 1 mM α-ketoglutarate in the cytosolic bath. c Summary of the results from similar experiments as shown in a and b. Notice that the cytosolic glutamate or its metabolite caused activation of gCFTR that is almost similar in magnitude. The presence of glutamate prevents α-toxin-induced deactivation of CFTR in the complete absence of ATP in the cytosol, indicating that glutamate activation of CFTR is independent of phosphorylation, as previously reported (Reddy and Quinton 2003). (C) and (L) represent cytosolic bath and lumen, respectively. The data represent mean ± SE

Fig. 6

Effect of cytosolic monovalent cations on phosphatase dephosphorylation of CFTR. a A representative electrical trace showing the effect of K⁺ substitution with Na⁺ on cAMP and G protein-activated CFTR. These experiments show that phosphorylation activation of CFTR-Cl⁻ conductance by cAMP and G proteins is sensitive to changes in cytosolic monovalent cation composition. That is, replacing cytosolic K⁺ with Na⁺ promptly deactivates gCFTR regardless of the mode of stimulation by cAMP or G proteins. G proteins are stimulated by GTP-γ-S. b A representative experiment showing the effect of cytoplasmic K⁺ substitution by Na⁺ on cAMP-activated CFTR after application of PP2A inhibitor okadaic acid. Notice that, unlike in Fig. 5a, cAMP can activate CFTR even after complete K⁺ substitution with Na⁺ after inhibiting endogenous PP2A with okadaic acid. These results show that okadaic acid-sensitive phosphatase dephosphorylates cAMP-phosphorylated CFTR as previously reported. c A representative experiment showing the effect of cytoplasmic K⁺ substitution by Na⁺ on G protein-activated CFTR after application of the PP2A inhibitor okadaic acid. Notice that, unlike cAMP, G protein activation with GTP-γ-S failed to stimulate CFTR after complete K⁺ substitution with Na⁺ even in the presence of okadaic acid. These results show that G protein-activated CFTR is dephosphorylated by an okadaic acid-insensitive phosphatase. (C) and (L) represent cytosolic bath and lumen, respectively

Fig. 7

Effect of okadaic acid on cAMP and G protein-activated gCFTR. Summary of the results from similar experiments as shown in Fig. 5. The data represent mean ± SE. * Significant difference from respective control values: P > 0.001

Fig. 8

Inconsistent cGMP regulation of CFTR. Unlike cAMP and G proteins, cGMP activation of CFTR is labile. The magnitude of activation of CFTR in sweat duct varied between the ducts and even within the same duct as a function of time. In some freshly isolated ducts the magnitude of cGMP activation of CFTR was comparable to that of cAMP (as shown in a), whereas in some other ducts the response is completely absent (as shown in b). cGMP stimulated gCFTR only in ~45% of the ducts, while cAMP and G proteins stimulated CFTR in almost 100% of the sweat ducts (c). (C) and (L) represent cytosolic bath and lumen, respectively

Fig. 9

Effect of cytosolic monovalent cations and okadaic acid on cGMP activation of gCFTR. a A representative electrical trace showing the effect of K⁺ substitution with Na⁺ on cGMP and activated CFTR. These experiments show that phosphorylation activation of CFTR-Cl⁻ conductance by cGMP is sensitive to changes in cytosolic monovalent cation composition. That is, replacing cytosolic K⁺ with Na⁺ promptly deactivates cGMP-activated gCFTR. b The data show that okadaic acid has no effect on the inhibition of gCFTR following cytosolic K⁺ substitution with Na⁺. These results indicate that, unlike PKA (cAMP)-phosphorylated CFTR, which is dephosphorylated by okadaic acid-sensitive phosphatase (Figs. 5, 6), PKG (cGMP)-phosphorylated CFTR is dephosphorylated by an okadaic acid-insensitive phosphatase, which is also sensitive to cytosolic changes in monovalent cations. (C) and (L) represent cytosolic bath and lumen, respectively