The long-term aim is to understand the mechanisms of regulation for transepithelial ion transport, using the epithelium of Necturus gallbladder as a model system for epithelia that transport NaC1 and water in isosmotic proportions. We will assess the function of apical and basolateral membrane ion channels, carriers and pumps, their regulation, and their functional interrelationships. With this general strategy in mind, we will: 1. Characterize apical membrane maxi K+ channels and baseline channels, emphasizing the effects of intracellular pH, which could contribute to regulating K+ secretion. 2. Characterize the apical membrane Cl-channel activated by cAMP and study the mechanisms of the effects of cAMP on the channel and the exchangers (phosphorylation/dephosphorylation, insertion of channels by exocytosis, removal of carriers by endocytosis). 3. Test whether pHi effects on allosteric titratable sites adjust the steady-state rates of the apical membrane Na+/H+ and Cl- /HCO3 - exchangers. 4. Characterize basolateral membrane K+ and Cl - channels and study the role of permanent buffer systems (HCO3 -/CO2, short-chain fatty acids) in stimulating basolateral membranes, in particular whether there is basolateral Na+/Ca2+ exchange, and assess the messenger roles of intracellular Ca2+ during experimental perturbations that alter ion transport. 6. Study the mechanisms of adjustment of the rates of apical membrane NaC1 entry and basolateral membrane ion transport, which must involve functional changes in the Na+ pump, the KC1 cotransporter and the K+ and Cl-channels. We will distinguish thermodynamic and cotransporter and the Cl channels. We will distinguish thermodynamic and kinetic factors contributing to these relationships and attempt to identify the cellular mechanisms involved. The gallbladder epithelium of Necturus maculosus has the advantages of structural simplicity (monolayered, flat epithelium, with one cell type and few microvilli), relatively large cells, and simple transepithelial ion and water transport mechanisms. We will address the issues raised above with a combination of electrophysiologic techniques (patch clamp, intra- and extracellular conventional and ion-selective microelectrodes), optical methods (measurements of intracellular pCa with fluorescent dyes) and ultrastructural techniques (electron microscopy and immunocytochemistry).
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