The broad, long term objectives of this proposal are to define the mechanisms of regulation of gastric HC1 secretion. Therapeutic control of gastric ulcer disease relies upon interventions which control HC1 secretion. Whereas the Mg2+-dependent, K+ dependent, and H+ transporting gastric H/K ATPase (E.C. 1.2.1.36) is responsible for the production of HC1 at the expense of ATP, little is known of the physiologically relevant mechanisms of control of HC1 secretion. As our major working hypothesis, the activity of Cl- channels of the secretory membrane of the parietal cell is a major target of regulation of HC1 secretion in mammals. We have recently achieved a breakthrough which allows a rigorous test of this hypothesis: Single channel recordings of Cl- channels of gastric vesicles fused to planar lipid bilayers have been obtained. Vesicles were prepared from the gastric mucosa of histamine treated rabbits. These vesicles are enriched in H/K ATPase. Upon stimulation, the H/K ATPase is maximally associated with the secretory membrane. These vesicle preparations are therefore enriched in secretory membranes, and exhibit MgATP, K+ and Cl-dependent HC1 accumulation and K+ and Cl- transport which are characteristic of the stimulated state of HC1 secretion. According to our major hypothesis, these vesicles should contain regulated Cl- channels. The key question is whether any of these represent the proposed stimulus-associated Cl- channel. Regardless of the technique used to measure single Cl- channels, whether patch clamp or planar lipid bilayers, it is necessary to demonstrate that the recorded channels arise from the cell and membrane of interest, and that the channel participates in the secretory process of interest. Fortunately, the rabbit parietal cell provides a uniquely useful system to relate the physiology, pharmacology, and biochemistry of macroscopic measures of secretory function such as HC1 secretion and net KC1 flux at the level of the cell and vesicle to recorded single channels.
The Specific Aims are 1) Cl- channels observed in single channel recordings in planar lipid bilayers will be characterized according to single channel unit conductance, voltage dependence, gating charge, selectivity for anions and cations, and sensitivity to channel blockers and changes in pH. These single channel characteristics will be compared and contrasted with 2) characteristics of macroscopic measures of Cl- channel function such as HC1 accumulation and net KC1 flux by cells and vesicles. Net behavior and macroscopic measures of Cl- channel function will be employed to investigate the role of recorded single Cl- channels in regulated HC1 secretion. 3) Stimulus associated changes in single Cl-channel behavior will be correlated with stimulus dependent changes in macroscopic measures of Cl- channel function (HC1 accumulation and net KC1 flux) such as might be afforded by ionized free Ca2+, or effectors of protein kinase C, protein kinase A, or G-proteins. Preliminary results of inhibitor and pH sensitivity suggest that the recorded single Cl- channels may participate in HC1 accumulation.
| 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 |
| 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 |
| 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 |
| 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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