The purpose of this study was to determine the mechanism of action of SPI-0211 (lubiprostone), a novel bicyclic fatty acid in development for the treatment of bowel dysfunction. Adult rabbit intestine was shown to contain mRNA for ClC-2 using RT-PCR, Northern blot analysis, and in situ hybridization. T84 cells grown to confluence on permeable supports were shown to express ClC-2 channel protein in the apical membrane. SPI-0211 increased electrogenic Cl- transport across the apical membrane of T84 cells, with an EC50 of approximately 18 nM measured by short-circuit current (Isc) after permeabilization of the basolateral membrane with nystatin. SPI-0211 effects on Cl- currents were also measured by whole cell patch clamp using the human embryonic kidney (HEK)-293 cell line stably transfected with either recombinant human ClC-2 or recombinant human cystic fibrosis transmembrane regulator (CFTR). In these studies, SPI-0211 activated ClC-2 Cl- currents in a concentration-dependent manner, with an EC50 of approximately 17 nM, and had no effect in nontransfected HEK-293 cells. In contrast, SPI-0211 had no effect on CFTR Cl- channel currents measured in CFTR-transfected HEK-293 cells. Activation of ClC-2 by SPI-0211 was independent of PKA. Together, these studies demonstrate that SPI-0211 is a potent activator of ClC-2 Cl- channels and suggest a physiologically relevant role for ClC-2 Cl- channels in intestinal Cl- transport after SPI-0211 administration.

An HEK-293 cell line stably expressing the human recombinant ClC-2 Cl(-) channel was used in patch-clamp studies to study its regulation. The relative permeability P(x)/P(Cl) calculated from reversal potentials was I(-) > Cl(-) = NO(3)(-) = SCN(-)>/=Br(-). The absolute permeability calculated from conductance ratios was Cl(-) = Br(-) = NO(3)(-) >/= SCN(-) > I(-). The channel was activated by cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleic acid (C:18 cisDelta9), elaidic acid (C:18 transDelta9), arachidonic acid (AA; C:20 cisDelta5,8,11,14), and by inhibitors of AA metabolism, 5,8,11,14-eicosatetraynoic acid (ETYA; C:20 transDelta5,8,11,14), alpha-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and 2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2 Cl(-) channels were activated by a combination of forskolin plus IBMX and were inhibited by the cell-permeant myristoylated PKA inhibitor (mPKI). Channel activation by reduction of bath pH was increased by PKA and prevented by mPKI. AA activation of the ClC-2 Cl(-) channel was not inhibited by mPKI or staurosporine and was therefore independent of PKA or protein kinase C activation.

Human ClC-2 Cl Ϫ (hClC-2) 1 channels are activated by PKA, an activation that is prevented by treatment with a permeant PKA inhibitor, myristoylated protein kinase inhibitor, mPKI (1, 2). These Cl Ϫ channels are also activated by reduced external pH (pH o ), but this activation requires PKA (1-5). Arachidonic acid also activates hClC-2, but this activation is not inhibited by mPKI and therefore is independent of PKA (2). ClC-2 plays a role in Cl Ϫ transport by a variety of tissues (6), and low pH o and PKA regulation of ClC-2 may be of physiological relevance. The goal of these studies was to determine the structural basis for low pH o and PKA activation of human ClC-2 Cl Ϫ channels. Human ClC-2 contains seven potential phosphorylation sites in the C terminus, and two in the N terminus (Table I). All but two consensus sites (RRAT655 and RGET691 in human ClC-2 and RRQS651 and KRKS749 in rabbit ClC-2) are conserved in rat (6). Human and rabbit, but not rat, ClC-2 are activated by PKA (1-6). These sites are absent in rat ClC-2 (6), a channel that does not show PKA activation, although it has been shown to be phosphorylated without changes in function (7). The phosphorylation sites RRAT655 and RGET691 of human ClC-2 were the focus of the present studies.ClC-2 (human and rabbit) has been shown to be activated by PKA in planar lipid bilayer studies and in hClC-2-expressing HEK-293 cells treated with forskolin plus IBMX (2, 5). However, an unsuccessful attempt to activate 36 Cl transport in human IB3 cells containing hClC-2 has been reported (8), despite finding low pH o activation. Further studies of the relationship between low pH o and PKA activation of hClC-2 were warranted because low pH o activation was found to be dependent upon PKA activation (2). The present studies used sitedirected mutagenesis and functional assays in the presence and absence of phosphatase inhibitors and a PKA inhibitor, mPKI, to determine the structural basis for PKA activation and PKA-dependent low pH o activation of hClC-2.The site(s) of PKA activation of the hClC-2 Cl Ϫ channel is not known. Moreover, it is not known whether PKA activation of the channel at pH o 7.4 and at reduced pH o occur at the same site(s). Site-directed mutagenesis of hClC-2 was used to determine which of the sites (RRAT and RGET), or whether both, was important under these two different conditions. Wild-type and mutant hClC-2 Cl Ϫ channels were stably expressed in HEK-293 cells, and Cl Ϫ channel function was assessed by measuring whole cell Cl Ϫ currents in response to activation of PKA by forskolin and IBMX at pH o 7.4 and 6.0. Arachidonic acid, which activates hClC-2 in a PKA-independent manner (2), was used to determine whether the mutant channels were expressed and present in the plasma membrane. The effects of protein phosphatase inhibitors were used to determine whether PKA activation was sufficient to overcome the action of endogenous protein phosphatase. These studies delineate the structural basis for low pH o and PKA activation of hClC-2 and suggest a ...

It has been difficult to separate/identify the roles of ClC-2 and CFTR in Cl(-) transport studies. Using pharmacological agents, we aimed to differentiate functionally between ClC-2 and CFTR Cl(-) channel currents. Effects of CFTR inhibitor 172 (CFTRinh172), N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), and methadone were examined by whole cell patch clamp on Cl(-) currents in recombinant human ClC-2/human embryonic kidney 293 (ClC-2/HEK293) cells stably transformed with Epstein-Barr nuclear antigen 1 (hClC-2/293EBNA) and human CFTR/HEK293 (hCFTR/HEK293) cells and by short-circuit current (Isc) measurements in T84 cells. Lubiprostone and forskolin-IBMX were used as activators. CFTRinh172 inhibited forskolin-IBMX-stimulated recombinant human CFTR (hCFTR) and lubiprostone-stimulated recombinant human ClC-2 (hClC-2) Cl(-) currents in a concentration-dependent manner equipotently. DASU-02 inhibited forskolin-IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, but not lubiprostone-stimulated Cl(-) currents in hClC-2/293EBNA cells. In T84 cells with basolateral nystatin or 1-ethyl-2-benzimidazolinone (1-EBIO), lubiprostone-stimulated and forskolin-IBMX-cyclosporin A (FICA)-stimulated Isc components were observed. CFTRinh172 inhibited major portions of both components. DASU-02 had no effect on lubiprostone-stimulated Isc but partially inhibited FICA-stimulated Isc. T84 cells in which ClC-2 or CFTR was knocked down using siRNAs were constructed. T84 ClC-2 knockdown cells did not respond to lubiprostone but did respond to forskolin-IBMX in a methadone-insensitive, DASU-02-sensitive manner, indicating CFTR function. T84 CFTR knockdown cells responded separately to lubiprostone and forskolin-IBMX in a methadone-sensitive and DASU-02-insensitive manner, indicating ClC-2 function. Low lubiprostone concentrations activated ClC-2, but not CFTR, and both channels were activated by forskolin-IBMX but have different inhibitor sensitivities. Methadone, but not DASU-02, inhibited ClC-2. DASU-02, but not methadone, inhibited CFTR. In T84 cells, both ClC-2 and CFTR are present and likely play roles in Cl(-) secretion.

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl− channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl− currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl− currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl− currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl− currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl2-inhibited Cl− currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl− currents with half-maximal inhibition at 100 and 200–230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl− currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl− currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl− currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl− currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.