Using Time-Resolved Fluorometry to Study the Transport Cycle of a Na+-Coupled Phosphate Cotransporter

Type II Na+-coupled phosphate cotransporters (NaPi-IIb) catalyze electrogenic phosphate transport with a 3:1 Na:HPO42- stoichiometry. No 3-D structure is currently available and we employ indirect approaches to investigate structure-function relations. With zero phosphate, voltage steps applied to Xenopus oocytes expressing NaPi-IIb induce presteady-state charge movements attributable to empty carrier reorientation and Na+ interactions. This transporter dynamic readout is global and provides limited insight into the underlying conformational changes. These can also be studied using voltage clamp fluorometry (VCF) that allows investigation of local responses to voltage and substrate. We have applied VCF to identify regions that respond to changes in the fluorophore's microenvironment close to the labeled site (Virkki et al., 2006, JBC, 281). We now focus on the time dependence of fluorescence intensity changes (DF) in response to voltage steps with different [Na]. Cysteines were substituted at externally accessible linker regions and after labeling with the fluorophore (MTS-TAMRA), the mutants showed WT-like cotransport behavior so that we could interpret our data in the context of the dynamics during the normal transport cycle. At two sites, one located in a functionally important re-entrant loop and the other in an adjacent linker, the time constants of DF (tauF)increased with [Na], typically from 3ms to 7ms for depolarizing steps. In contrast, DF recorded from a site in the first extracellular linker showed a tauF independent of [Na]. This indicated that the fluorophore microenvironment at this site was sensitive to voltage-dependent conformation changes associated with the empty carrier only. In all cases, tauF was significantly slower than the simultaneously measured presteady-state charge relaxations, which suggested that these local changes follow the overall global movements. Our findings will be incorporated into a map of voltage- and substrate-dependent conformational changes.