title: Characterization of the human pH- and PKA-activated ClC-2G(2 alpha) Cl- channel.
abstract: 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.
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Ohio 45267-0576, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Sherry
authors.forename: K
authors.initials: K
authors.lastname: Stroffekova
authors.forename: L M
authors.initials: LM
authors.lastname: Knapp
authors.forename: E Y
authors.initials: EY
authors.lastname: Kupert
authors.forename: J
authors.initials: J
authors.lastname: Cuppoletti
authors.forename: D H
authors.initials: DH
authors.lastname: Malinowska
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002678:Chimera; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005333:Fetus; D005753:Gastric Mucosa / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000196:embryology / Q000378:metabolism; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D011817:Rabbits; D013270:Stomach / Q000166:cytology; D014982:Xenopus laevis
chemical_list: D018118:Chloride Channels; D018076:DNA, Complementary; D006859:Hydrogen; D017868:Cyclic AMP-Dependent Protein Kinases