Therapeutic control of gastric ulcer disease relies upon interventions which control HCl secretion. The broad, long term objectives of this proposal are to define the molecules and mechanisms involved in regulation of gastric HCl secretion. Whereas ATP fuels the Mg2+-dependent, K+- dependent and H+-transporting gastric H/K ATPase (EC 1.2.1.26), HCl production by the H/K ATPase also requires K+ and Cl- passage from the cytosol across the apical membrane to the secretory canaliculus. K+ is a substrate for the H/K ATPase and Cl- provides equivalents of Cl- for HCl production. Ion transport across the gastric parietal cell apical membrane is under control by cAMP dependent protein kinase. These channels function under the harsh, but physiologically relevant condition of pH 3 or lower bathing the extracytosolic face of the channel. An acid and voltage activated Cl- channel has been characterized, cloned and expressed, and an acid stable K+ channel has also been characterized during the past 3 year funding period. The channels have been shown to be targets, per se, of cAMP dependent protein kinase. In vitro activation of HCl accumulation by isolated gastric vesicles has also been demonstrated. The goals of the present proposal are to further elucidate the role of these newly discovered Cl- and K+ channels of the gastric parietal cell secretory membrane in regulated HCl secretion. The proposed studies are essential to understanding the physiologically relevant mechanisms of control of HCl secretion. As the major working hypothesis, the activity of the ion channels of the secretory membrane of the parietal cell represent a major target of the regulation of HCl secretion in mammals. The focus of the present application is directed toward understanding how these ion channels function in regulated HCl secretion.
The specific aims are therefore to l) Compare and contrast the electrophysiological properties of the channels with macroscopic measures of K+ and Cl- flux and H+ accumulation. 2) Determine the mechanisms of regulation of the acid and voltage regulated Cl- channel from native and recombinant sources. Regulation of the newly identified K+ channel will also be studied 3) Determine the structural basis for the unique regulatory aspects of the Cl- channel. 4) Determine the sites of localization of the Cl- channel to cells within the gastric mucosa, and the distribution of the Cl- channels within the parietal cell in the non-secreting and secreting state and; 5) Examine the hypothesis that the gastric Cl- channel is encoded by an alternatively spliced transcript of the C1C-2 gene. These studies provide important molecular and mechanistic links with physiological cellular and whole animal studies of HCl secretion and may provide a paradigm for understanding the cellular and molecular mechanisms which underlie regulated secretion by epithelia.
| Nighot, Meghali P; Nighot, Prashant K; Ma, Thomas Y et al. (2015) Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion. PLoS One 10:e0138174 |
| Cuppoletti, John; Malinowska, Danuta H; Tewari, Kirti P et al. (2004) SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents. Am J Physiol Cell Physiol 287:C1173-83 |
| Cuppoletti, John; Tewari, Kirti P; Sherry, Ann M et al. (2004) Sites of protein kinase A activation of the human ClC-2 Cl(-) channel. J Biol Chem 279:21849-56 |
| Malinowska, Danuta H; Sherry, Ann M; Tewari, Kirti P et al. (2004) Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+ channels. Am J Physiol Cell Physiol 286:C495-506 |
| Cuppoletti, J; Tewari, K P; Sherry, A M et al. (2001) ClC-2 Cl- channels in human lung epithelia: activation by arachidonic acid, amidation, and acid-activated omeprazole. Am J Physiol Cell Physiol 281:C46-54 |
| Sherry, A M; Malinowska, D H; Morris, R E et al. (2001) Localization of ClC-2 Cl- channels in rabbit gastric mucosa. Am J Physiol Cell Physiol 280:C1599-606 |
| Tewari, K P; Malinowska, D H; Sherry, A M et al. (2000) PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels. Am J Physiol Cell Physiol 279:C40-50 |
| Stroffekova, K; Kupert, E Y; Malinowska, D H et al. (1998) Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel. Am J Physiol 275:C1113-23 |
| Sherry, A M; Stroffekova, K; Knapp, L M et al. (1997) Characterization of the human pH- and PKA-activated ClC-2G(2 alpha) Cl- channel. Am J Physiol 273:C384-93 |
| Huang, P; Stroffekova, K; Cuppoletti, J et al. (1996) Functional expression of the cystic fibrosis transmembrane conductance regulator in yeast. Biochim Biophys Acta 1281:80-90 |
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