Cysteine mutagenesis reveals novel structure-function features within the predicted third extracellular loop of the type IIa Na+/P-i cotransporter

The transport function of the rat type IIa Na+/P-j cotransporter is inhibit ed after binding the cysteine modifying reagent 2-aminoethyl methanethiosul fonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This sug gests that Ser-460 lies in a functionally important region of the protein. To establish a "structure-function" profile for the regions that flank Ser- 460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-46 5 were substituted one by one for a cysteine. Mutants were expressed in Xen opus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cys teine modifying (methanethiasulfonate, MTS) reagents. Except for mutant 144 7C, mutants with cysteines at sites from Arg-437 through Thr-449, as well a s Pro-461, were inactive. Cotransport function of mutants with Cys substitu tions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagen ts. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an a-helix motif . Apart from loss of transport function, exposure of mutants A453C and A455 C to MTSEA or 2-(triethylammonium) ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway Mutants from Ala-453 through Ala-459 showed less PPI dependency, bu t generally stronger voltage dependency compared with the wild type, wherea s those flanking this group were more sensitive to pH and showed weaker vol tage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fu lly loaded carrier and show a membrane-associated alpha -helical structure.