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.

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

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 ...

HCl secretion across the parietal cell apical secretory membrane involves the H+-K+-ATPase, the ClC-2 Cl- channel, and a K+ channel. In the present study, the cellular and subcellular distribution of ClC-2 mRNA and protein was determined in the rabbit gastric mucosa and in isolated gastric glands. ClC-2 mRNA was localized to parietal cells by in situ hybridization and by direct in situ RT-PCR. By immunoperoxidase microscopy, ClC-2 protein was concentrated in parietal cells. Immunofluorescent confocal microscopy suggested that the ClC-2 was localized to the secretory canalicular membrane of stimulated parietal cells and to intracellular structures of resting parietal cells. Immunogold electron microscopy confirmed that ClC-2 is in the secretory canalicular membrane of stimulated cells and in tubulovesicles of resting parietal cells. These findings, together with previous functional characterization of the native and recombinant channel, strongly indicate that ClC-2 is the Cl- channel, which together with the H+-K+-ATPase and a K+ channel, results in HCl secretion across the parietal cell secretory membrane.