The broad, long-term objectives of this proposal are to determine the mechanism(s) responsible for the alanine-stimulated increase in enterocyte plasma membrane K conductance (Gk) and if the activated Gk contributes to regulate of enterocyte volume. Alanine-induced increases in apical membrane exocytosis and capacitance suggest that the increase in plasma membrane Gk in Aplysia enterocytes, a model nutrient absorbing epithelium, results from exocytic insertion of K channels into the apical membrane. Hence, the specific aims are to: 1) assess the effect of inhibitory G proteins (Gi) are necessary for the alanine-induced increases in exocytosis and membrane capacitance; 3) determine the effect of alanine on enterocyte volume and if the activated Gk is necessary for regulation of enterocyte volume; and 4) assess the effect of alanine on the relative number of apical membrane K channels. The research design is to: 1) assess the effect of alanine on translocation of fluorescently-labeled vesicles to the apical plasma membrane, 2) determine whether microtubules and G proteins are necessary for exocytic insertion of K channels into the apical membrane with microtubule inhibitors and AIF/4 which activates heterotrimeric G proteins, 3) determine the effect of alanine on enterocyte volume; and 4) assess the effect of alanine on the relative number of apical membrane K channels. The research membrane, 2) determine whether microtubules and G proteins and G proteins are necessary for exocytic insertion of K channels into the apical membrane with microtubule inhibitors and AIF4 which activates heterotrimeric G proteins, 3) determine the effect of alanine on enterocyte volume; and 4) assess the effect of 10 nM mannitol (control) and 10 mM alanine (treatment) on number of K channels in the apical membrane. The methods to be used are: 1) fluorescence microscopy to visualize translocation of intracellular vesicles to the apical membrane; 2) a microelectrode technique to determine capacitance of the apical membrane; 3) a fluid-phase marker, horseradish peroxidase, to determine the rate of exocytosis; 4) an electrical resistance method to determine the effect of alanine on enterocyte volume; and 5) cell surface biotinylation and Western blot analysis to determine the relative number of apical membrane K channels. The health-relatedness of the project is that it may reveal a strategy to increase salt and water absorption from the digestive tract in individuals suffering from severe dehydration. Strategies designed to inhibit Gi proteins involved in modulating exocytic insertion of K channels into plasma membranes could increase delivery of channels to the cell surface. This could increase the driving force for Na/nutrient co-transport and stimulate absorption of solutes and water from the digestive tract and enhance the rate of rehydration of those individuals suffering from dehydration. These studies will provide important information about regulation of the pump-leak model that generates membrane potentials and controls cell volume in nutrient- stimulated Na-absorbing epithelia.
| Keeton, Robert Aaron; Runge, Steven William; Moran, William Michael (2004) Constitutive apical membrane recycling in Aplysia enterocytes. J Exp Zool A Comp Exp Biol 301:857-66 |
| Keeton, R A; Runge, S W; Moran, W M (2004) Alanine-stimulated exocytosis in Aplysia enterocytes: effect of Na+ transport and requirement for actin filaments. J Comp Physiol B 174:129-38 |
