Porous membranes for reconstitution of ion channels.

Functional biological synthetic composite (BSC) membranes were made using phospholipids, biological membrane proteins and permeable synthetic supports or membranes. Lipid bilayers were formed on porous polycarbonate (PC), polyethylene terephthalate (PETE) and poly (l-lactic acid) (PLLA) membranes and in 10-100 microm laser-drilled pores in a 96-well plastic plate as measured by increased resistance or decreased currents. Bilayers in 50 microm and smaller pores were stable for up to 4 h as measured by resistance changes or a current after gramicidin D reconstitution. Biological membrane transport reconstitution was then carried out. Using vesicles containing Kv1.5 K(+) channels, K(+) currents and decreased resistance were measured across bilayers in 50 microm pores in the plastic plate and PLLA membranes, respectively, which were inhibited by compound B, a Kv1.5 K(+) channel inhibitor. Functional reconstitution of Kv1.5 K(+) channels was successful. Incorporation of membrane proteins in functional form in stable permeable membrane-supported lipid bilayers is a simple technology to create BSC membranes that mimic biological function which is readily adaptable for high throughput screening.

ClC-2 Cl- channels in human lung epithelia: activation by arachidonic acid, amidation, and acid-activated omeprazole.

ClC-2 Cl- channels represent a potential target for therapy in cystic fibrosis. Key questions regarding the feasibility of using ClC-2 as a therapeutic target are addressed in the present studies, including whether the channels are present in human lung epithelia and whether activators of the channel can be identified. Two new mechanisms of activation of human recombinant ClC-2 Cl- channels expressed in HEK-293 cells were identified: amidation with glycine methyl ester catalyzed by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and treatment with acid-activated omeprazole. ClC-2 mRNA was detected by RT-PCR. Channel function was assessed by measuring Cl- currents by patch clamp in the presence of a cAMP-dependent protein kinase (PKA) inhibitor, myristoylated protein kinase inhibitor, to prevent PKA-activated Cl- currents. Calu-3, A549, and BEAS-2B cell lines derived from different human lung epithelia contained ClC-2 mRNA, and Cl- currents were increased by amidation, acid-activated omeprazole, and arachidonic acid. Similar results were obtained with buccal cells from healthy individuals and cystic fibrosis patients. The ClC-2 Cl- channel is thus a potential target for therapy in cystic fibrosis.

Localization of ClC-2 Cl- channels in rabbit gastric mucosa.

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.

Activation of human CIC-2 Cl- channels: implications for cystic fibrosis.

1. The CIC-2 Cl- channels are present in the adult human lung epithelia and, therefore, are a potential target for therapy in cystic fibrosis. 2. Activators of CIC-2 Cl- channels that may have physiological relevance include activation by reduced external pH, protein kinase A and arachidonic acid. 3. Activators of CIC-2 Cl- channels that have therapeutic potential include amidation and omeprazole and, perhaps, effectors of arachidonic acid metabolism.

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels.

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.

Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel.

Rabbit and human ClC-2G Cl- channels are voltage sensitive and activated by protein kinase A and low extracellular pH. The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl- channel and to determine which amino acid residues play a role in this acid activation. Channel open probability (Po) at +/-80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+ concentration (that is, extracellular pH, pHtrans), with an apparent acidic dissociation constant of pH 4.95 +/- 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased Po threefold at pHtrans 7.4, at which the channel normally exhibits low Po. With extracellular pH reduction (protonation) or amidation, increased Po was due to a significant increase in open time constants and a significant decrease in closed time constants of the channel gating, and this effect was insensitive to applied voltage. With the use of site-directed mutagenesis, the extracellular region EELE (amino acids 416-419) was identified as the pH sensor and amino acid Glu-419 was found to play the key or predominant role in activation of the ClC-2G Cl- channel by extracellular acid.

Characterization of the human pH- and PKA-activated ClC-2G(2 alpha) Cl- channel.

A ClC-2G(2 alpha) Cl- channel was identified to be present in human lung and stomach, and a partial cDNA for this Cl- channel was cloned from a human fetal lung library. A full-length expressible human ClC-2G(2 alpha) cDNA was constructed by ligation of mutagenized expressible rabbit ClC-2G(2 alpha) cDNA with the human lung ClC-2G(2 alpha) cDNA, expressed in oocytes, and characterized at the single-channel level. Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) treatment increased the probability of opening of the channel (Po). After PKA activation, the channel exhibited a linear (r = 0.99) current-voltage curve with a slope conductance of 22.1 +/- 0.8 pS in symmetric 800 mM tetraethylammonium chloride (TEACl; pH 7.4). Under fivefold gradient conditions of TEACl, a reversal potential of +21.5 +/- 2.8 mV was measured demonstrating anion-to-cation discrimination. As previously demonstrated for the rabbit ClC-2G(2 alpha) Cl- channel, the human analog, hClC-2G(2 alpha), was active at pH 7.4 as well as when the pH of the extracellular face of the channel (trans side of the bilayer; pHtrans) was asymmetrically reduced to pH 3.0. The extent of PKA activation was dependent on pHtrans. With PKA treatment, Po increased fourfold with a pHtrans of 7.4 and eightfold with a pHtrans of 3.0. Effects of sequential PKA addition followed by pHtrans reduction on the same channel suggested that the PKA- and pH-dependent increases in channel Po were separable and cumulative. Northern analysis showed ClC-2G(2 alpha) mRNA to be present in human adult and fetal lung and adult stomach, and quantitative reverse transcriptase-polymerase chain reaction showed this channel to be present in the adult human lung and stomach at about one-half the level found in fetal lung. The findings of the present study suggest that the ClC-2G(2 alpha) Cl- channel may play an important role in Cl- transport in the fetal and adult human lung.

Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.

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.

Differential acidic pH sensitivity of delta F508 CFTR Cl- channel activity in lipid bilayers.

Cystic fibrosis transmembrane conductance regulator (CFTR) is present in acidic intracellular vesicles. Human normal and delta F508 CFTR Cl- channel characteristics at pH 7.4 and pH 4.5 were determined by fusing Xenopus laevis oocyte plasma membranes containing the expressed channels to planar lipid bilayers. At pH 7.4, both channels exhibited linear current-voltage curves, a 10 +/- 0.3-pS conductance using 800 mM CsCl, and a 9:1 Cl-/Cs+ discrimination ratio obtained from a 32 +/- 2 mV reversal potential with a fivefold gradient. At -80 mV, the open probability (Po) of mutant CFTR was 53% that of normal CFTR. Reduction of the trans-pH from 7.4 to 4.5 had no effect on the above characteristics except for Po, where it caused a 47% reduction in normal CFTR Po (due to a 75% decrease in mean open time) and a 75% reduction in delta F508 CFTR Po (due to a 6-fold increase in mean closed time). Normal CFTR can thus function in the environment of acidic intracellular organelles, whereas activity of mutant CFTR would be greatly reduced. These results may be of significance to understanding the cystic fibrosis defect.

Cl- channels of the gastric parietal cell that are active at low pH.

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulus-associated protein in gastric parietal cell detected using antimelittin antibody.

The bee venom polypeptide melittin binds to and inhibits the gastric hydrogen-potassium-adenosinetriphosphatase (H(+)-K(+)-ATPase). A search for parietal cell proteins with a melittin-like structure was carried out. A 67-kDa (doublet) protein, which reacted with a polyclonal antimelittin antibody, was found in purified rabbit parietal cells. The protein exhibited reversible stimulus-dependent redistribution from cytosol to (total) membranes. It was also found to be associated with H(+)-K(+)-ATPase-containing membranes when isolated from the gastric mucosae of rabbits treated with histamine, but not with cimetidine. The presence of the protein correlated with the ability of the membrane preparations to exhibit ionophore-independent HCl accumulation, a characteristic of gastric membranes from histamine-stimulated animals. The 67-kDa melittin-like protein may play a role in the functional changes in the gastric parietal cell that are involved in stimulation of HCl secretion.

Interaction of polypeptides with the gastric (H+ + K+)ATPase: melittin, synthetic analogs, and a potential intracellular regulatory protein.

The 26 amino acid bee venom toxin, melittin, is an amphipathic helical polypeptide which inhibits the gastric (H+ + K+)ATPase. The site of interaction with the (H+ + K+)ATPase was shown to be the alpha subunit of the (H+ + K+)ATPase in studies using [125I]azidosalicylyl melittin, a radioactive photoaffinity analog of melittin. A synthetic amphipathic polypeptide (Trp3) containing tryptophan, which exhibits a structure similar to that of melittin, also inhibited the gastric (H+ + K+)ATPase, and prevented labeling by [125I]azidosalicylyl melittin. These findings suggested that melittin and the synthetic amphipathic helical polypeptide were bound to the same or overlapping site(s). In the present studies, novel tritiated photoaffinity analogs of Trp3 containing benzoylphenylalanine (in place of tryptophan) were used to photoaffinity label the (H+ + K+)ATPase. These studies help to establish that the (H+ + K+)ATPase contains a binding site for polypeptides which exhibit an amphipathic helical motif. The precise amino acid sequence of the polypeptide appears to be of secondary importance for interaction with the (H+ + K+)ATPase as long as the alpha helical motif is present. The benzoylphenylalanine containing polypeptides are ideal for mapping the binding site on the (H+ + K+)ATPase. Using an antibody which recognizes this amphipathic helical ('melittin-like') motif, we have demonstrated that the gastric parietal cell contains a 67 kDa 'melittin-like' protein. This protein was associated with the gastric parietal cell apical membrane in the stimulated (secreting) state, but not in the resting (non-secreting) state. The binding site for the gastric 'melittin-like' protein appears to overlap with the melittin binding site on the alpha subunit of the (H+ + K+)ATPase. The potential physiological significance of the melittin binding site and the overlapping binding site for this newly identified endogenous 'melittin-like' protein on the (H+ + K+)ATPase to regulated HCl secretion by the parietal cell is presently under investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cl- channel blockers inhibit acid secretion in rabbit parietal cells.

Mechanisms of gastric parietal cell secretory membrane Cl- transport and the role of this Cl- transport in acid secretion were investigated by examining the effects of two Cl- channel blockers, diphenylamine-2-carboxylate (DPC) and 9-anthracene carboxylate (9-AC) on acid secretion using isolated, enriched rabbit parietal cells. Resting and stimulated acid secretion in intact cells (measured as [14C]aminopyrine accumulation) was inhibited by DPC and 9-AC, irrespective of agonist used. Apparent inhibition constants (Ki) were 2.4 x 10(-4) M for DPC and 1.2 x 10(-3) M for 9-AC for all responses. Digitonin-permeabilized parietal cells were used to bypass possible inhibitory effects of these compounds on basolateral membrane transport processes and to investigate effects only on the secretory membrane. Both blockers inhibited ATP-driven acid secretion in resting and stimulated permeable cells with apparent Ki values in the same range as measured in intact cells, suggesting that the site of action of these blockers is at the secretory membrane. H(+)-K(+)-ATPase activity in situ in permeable parietal cells, measured as 2-methyl-8-(phenylmethoxy)imidazo(1,2) pyridine-3-acetonitrile (SCH28080)-inhibitable ATP hydrolysis, was higher in stimulated compared with resting cells. Addition of 10 mM NH4Cl abolished this difference, and maximal H(+)-K(+)-ATPase activity was measured. SCH28080 and NH4Cl each abolished both resting and stimulated acid accumulation. DPC and 9-AC inhibited resting and stimulated H(+)-K(+)-ATPase activities, without exerting inhibitory effects on the enzyme itself, since the blockers had no effect on maximal NH4(+)-stimulated H(+)-K(+)-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin.

Melittin is a 26-amino acid amphipathic polypeptide toxin from bee venom which forms anion-selective ion channels in bilayers and biological membranes under the influence of membrane potential. Melittin has been shown to interact with a number of membrane proteins. We found that melittin inhibited K+-stimulated ATP hydrolysis by the (H+ + K+) ATPase in parietal cell apical membrane vesicles derived from histamine-stimulated rabbit gastric mucosa with a KIapp of 0.5 micron. Melittin also inhibited K+-stimulated p-nitrophenyl hydrolysis activity which is associated with the gastric (H+ + K+) ATPase in a dose-dependent manner with a KIapp of 0.95 micron. ATP-driven, K+-dependent H+ transport was inhibited over this same concentration range, even in the absence of a membrane potential. Melittin did not appear to increase the H+ leak from vesicle with preformed H+ gradients when the H+ pump was arrested by Mg2+ chelation, but all possible membrane perturbation effects were difficult to rule out. However, the data suggest that melittin exerts its inhibitory effect through interaction with the (H+ + K+) ATPase. In order to determine whether direct interactions between the (H+ + K+) ATPase and melittin occurred, a radioactive derivative of melittin, [125I]azidosalicylyl melittin, was prepared and photoreacted with sealed rabbit gastric membranes and highly purified hog gastric membrane containing the (H+ + K+) ATPase. In the purified hog preparation only a 95,000-Da band, the (H+ + K+) ATPase was labeled, while in the rabbit preparation a 95,000-Da band and one other membrane protein of 70,000 Da were labeled with this reagent. Label incorporation into the (H+ + K+) ATPase and the 70,000-Da band was greatly reduced by addition of excess unlabeled melittin, suggesting specificity of the interaction. Label incorporation occurred in the absence of ATP or added salts and was not reduced by SCH28080 (a K+ site inhibitor) suggesting that the melittin binding site was distinct from the luminal K+ site of action of SCH28080.

Gastric H+ secretion: histamine (cAMP-mediated) activation of protein phosphorylation.

Activation of H+ secretion by the gastric parietal cell involves major changes in morphology, metabolic activity and ion pathways of the secretory membrane. These changes are elicited by histamine binding to the H2 receptor, raising cAMP levels and presumably activating cAMP-dependent protein kinase. Concomitantly, the intracellular free Ca2+ concentration, [Ca2+]i, increases. Studies were performed to determine whether cAMP-mediated protein phosphorylation accompanies histamine activation of H+ secretion and to catalogue the major protein species serving as substrates for cAMP-dependent protein kinase in the parietal cell. 80% pure rabbit parietal cells, prepared by Nycodenz bouyant density centrifugation, were used. To investigate only cAMP-mediated effects, histamine-dependent changes in [Ca2+]i in these cells were abolished by depleting intracellular Ca2+ stores and performing experiments under Ca2+-free conditions. Acid secretion and steady-state levels of protein phosphorylation were then measured in unstimulated (cimetidine-treated) and histamine-stimulated cells. In intact parietal cells, concommitant with histamine stimulation of H+ secretion, increases in the level of protein phosphorylation were observed. Significantly changing phosphoproteins found in supernatant fractions showed apparent subunit sizes of approx. 148, 130, 47 and 43 kDa, and in microsomal fractions included those at approx. 130, 51 and 47 kDa. In parietal cell homogenates, using [gamma-32P]ATP, cAMP elicited significant phosphorylation of eight supernatant proteins and twelve microsomal proteins, which included the histamine-dependent phosphoproteins found in the intact parietal cell, except for the 51 kDa microsomal protein. As a working hypothesis, these proteins are involved in stimulus-secretion coupling in the parietal cell.

Cytoskeletal determinants of control of gastric acid secretion.

Isolated purified parietal cells from rabbit gastric mucosae were used to investigate the functions of phosphoproteins associated with regulation of acid secretion. These cells responded well to secretagogues, increasing their level of acid secretion by ca. four-fold. Stimulation of acid secretion was accompanied by a distinct morphological change, which involves the cytoskeletal system of the parietal cell, particularly the microfilaments, as evidenced by potent inhibition of secretion by cytochalasin E. Parietal cells contained several actin binding proteins, at least one of which (47 kD) had a similar molecular weight as a previously identified stimulus-related phosphoprotein. A single 130 kD parietal cell protein was immunoreactive with anti-vinculin monoclonal antibody, correlating with another previously identified stimulus-related phosphoprotein. The functional role of these proteins may be to link the microfilaments with the secretory membrane, an important step in the generation and/or maintenance of the stimulated state of acid secretion in the parietal cell.