We have applied voltage-clamp fluorometry, which combines two-electrode voltage clamp with fluorescence measurements, to detect conformational changes of the type IIb Na⁺-coupled P_i cotransporter, expressed in Xenopus oocytes. We introduced novel Cys at three sites in the protein and recorded voltage-and substrate-dependent changes in fluorescence ([Delta]F). Neither mutation nor labelling changed significantly the electrogenic properties of the mutants. Different [Delta]F responses to voltage and substrate were recorded at the three sites. S155C, predicted to lie in a re-entrant loop, showed strongly Na⁺, Li⁺ -and P_i-dependent [Delta]F signals, with increased quench of fluorescence seen at negative pontetials. In contrast, S226C and Q319C, located at opposite ends of a large extracellular loop, mainly responded to changes in Na⁺ and Li⁺. Interestingly, although Na⁺ and Li⁺ affected [Delta]F similarly in the two mutants, their voltage dependencies were complementary: For S226C F was quenched by hyperpolarization, and for Q319C by depolarization.

For S155C, the result confirms the re-entrant nature of this loop, as well as its involvement in conformational changes during the transport cycle of the protein. The data for S226C and Q319C are the first to show involvement of this loop in transport function. Their reciprocal DF voltage-dependencies suggest that these two sites are subjected to similar but opposite conformational movements during the transport cycle.