Isotype-specific Activation of Cystic Fibrosis Transmembrane Conductance Regulator-Chloride Channels by cGMP-dependent Protein Kinase II

Abstract

Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type I␣ cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl ؊ channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl ؊ channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl ؊ channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATP␥S (adenosine 5-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKI␣, is a potential activator of chloride transport in CFTR-expressing cell types.

cGMP Stimulation of Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels Co-expressed with cGMP-dependent Protein Kinase Type II but Not Type Iβ

“…In excised membrane patches, the activation of CFTR Cl Ϫ channels by purified pig cGK II was relatively slow in comparison to their activation by cAK (13). In Ad-cGK II-infected IEC-CF7 cells 8-pCPT-cGMP caused a similar sluggish activation of CFTR ( Fig.…”

“…Furthermore, purified endogenous pig cGK II, in contrast to bovine lung cGK I, was shown to activate CFTR in excised membrane patches (13). However, the mechanism of the apparent cGK isotype selectivity in activating CFTR was not clear, since both cGK I and cGK II could phosphorylate immunoprecipitated CFTR in vitro (13). An explanation for this discrepancy might be that cGK II but not cGK I selectively phosphorylates CFTR in a native environment.…”

“…In contrast, type I cGK, consisting of ␣ and ␤ isoforms, and shown to act as a key regulator of cardiovascular homeostasis (11,12), is not expressed in enterocytes (7). Furthermore, purified endogenous pig cGK II, in contrast to bovine lung cGK I, was shown to activate CFTR in excised membrane patches (13). However, the mechanism of the apparent cGK isotype selectivity in activating CFTR was not clear, since both cGK I and cGK II could phosphorylate immunoprecipitated CFTR in vitro (13).…”

“…Purification of the membrane-bound cGK II requires the use and subsequent removal of detergents, a procedure which potentially contributes to nonspecific (hydrophobic) interactions of this enzyme. Furthermore, purification results in partial proteolytic modification of cGK II and renders cGK II less sensitive to cGMP (5,13).…”

A cyclic nucleotide PDE5 inhibitor corrects defective mucin secretion in submandibular cells containing antibody directed against the cystic fibrosis transmembrane conductance regulator protein

“…Although cyclic GMP increases in response to the most potent stimulator of mucin secretion, isoproterenol, are small, it is possible that either cyclic AMP or cyclic GMP can activate CFTR to induce stimulation of mucin secretion. Thus, in addition to the well-established activation of CFTR by cyclic AMP-activated protein kinase [3], the type II, membrane-associated cyclic GMP-activated protein kinase has been shown to phosphorylate CFTR and activate Cl 3 transport in rat intestinal cells expressing CFTR [33]. In addition, although the C-type natriuretic peptide, which increased cyclic GMP levels in airways epithelial cells [34], did not stimulate ion transport in human cultured surface epithelial cells [35] it did increase Cl 3 transport in mouse nasal epithelium [36] and in a human airways gland cell line [37].…”