This proposal is aimed at evaluating the molecular mechanism by which membrane potentials and changes in membrane potential can regulate the function of Na+-coupled transport systems. It emphasizes a new conceptual perspective, the "Na+-well" model, which envisions the possibility that ion binding sites on the transport protein are "embedded" relatively deep in the membrane matrix and are reached via a channel-like access route that exhibits ion specificity, so that a Na+ ion will 'sense" a part of the electric field represented by the membrane potential in reaching or dissociating from the ion binding site. This, in turn, suggests that a primary role for the potential in governing transport capability is the result of potential-dependent Na+ binding/dissociation events associated with the transport cycle. This concept is an alternative to the usually considered "translocation" models in which the potential is envisioned as altering the transition rate between different carrier conformational states ("inward" vs. "outward" facing) for either the free or loaded carrier forms. Cultured LLC-PK1 cells are the only model system to be employed. Two primary experimental approaches are described. In one, whole-cell recording procedures will be used to monitor the function of Na+-coupled solute transport under conditions in which the two primary thermodynamic diving forces (Na+ gradient and membrane potential) are carefully controlled and the resulting kinetic effects on the full transport cycle can be measured. Experiments are described for evaluating the mechanistic role of potentials in two different Na+-coupled transport systems - for alpha-methylglucoside and alanine. These systems differ in Na+ coupling stoichiometry and therefore in the degree of complexity for modeling transport kinetics and the role that potentials play in governing function. In a second approach, isotope flux techniques will be used to measure the rate and degree of potential dependence for segments of the transport cycle for Na+-coupled systems under conditions where the full sequence of cycle events does not occur. The two approaches offer different insights to transport mechanism because whole cell recording measures net transport of charge catalyzed by the full transport cycle whereas isotope flux experiments offer high sensitivity for monitoring events that can be catalyzed by exchange loops in segments of the cycle. 14C-sugar and 22Na+ exchanges will be used to probe the rate and potential dependence for different segments in the full transport cycle and provide useful comparisons to related parameters when the full cycle is operative. Exchanges offer particular value for sensing potential dependent conformational changes in the full loaded sugar carrier. Non-exchanges mode isotope flux experiments and reactivity with chemical probes will be employed to determine if potential dependent conformational changes can be detected for the substrate-free carrier.