The transport function of the rat type IIa Na+/Pi cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests 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-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an α-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C 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 pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated α-helical structure.
Cysteine Mutagenesis Reveals Novel Structure–Function Features within the Predicted Third Extracellular Loop of the Type Iia Na+/Pi Cotransporter
Drs. Lambert and Forster contributed equally to this work and should be considered co-first authors.
Abbreviations used in this paper: ECL-3: third extracellular loop; MTS, methanethiosulfonate; MTSEA, 2-aminoethyl MTS hydrobromide; MTSET, 2-(triethylammonium)ethyl MTS bromide; NaPi-IIa, type IIa sodium phosphate cotransporter; PFA phosphonoformic acid; Pi, inorganic phosphate; SCAM, substituted cysteine accessibility method; TMD, transmembrane domain; WT, wild type.
A third possibility, that the number of active transporters in the membrane was partially reduced (for example, by the induction of endocytosis by the MTS reagent), could be rejected because we observed no change in membrane capacitance during the incubation period (data not shown). This would otherwise indicate a reduction of membrane area that normally accompanies endocytosis of NaPi-IIa (Forster et al. 1999b).
Michaelian responses ( with nH = 1.0) were obtained for nine mutants for which we determined Pi activation curves (Fig. 7 A, *), with KmPi ranging from 0.050 ± 0.001 to 0.12 ± 0.03 mM (n = 4), compared with 0.06 mM previously reported for the WT (Forster et al. 1998). For six mutants (Fig. 7 A, **) the Na+ activation data yielded KmNa estimates in the range 44 ± 7 to 98 ± 21 mM (n = 4), compared with 52 mM for the WT (Forster et al. 1998) and with Hill coefficients that were all significantly >2 (compare nHNa = 2.9 for the WT, Forster et al. 1998).
Georg Lambert, Ian C. Forster, Gerti Stange, Katja Köhler, Jürg Biber, Heini Murer; Cysteine Mutagenesis Reveals Novel Structure–Function Features within the Predicted Third Extracellular Loop of the Type Iia Na+/Pi Cotransporter. J Gen Physiol 1 June 2001; 117 (6): 533–546. doi: https://doi.org/10.1085/jgp.117.6.533
Download citation file:
Sign in
Client Account
Sign in via your Institution
Sign in via your InstitutionSuggested Content
Email alerts
Advertisement