Properties of the mutant ser-460-cys implicate this site in a functionallyimportant region of the type IIa Na+ /P-i cotransporter protein

The substituted cysteine accessibility approach, combined with chemical mod ification using membrane-impermeant alkylating reagents, was used to identi fy functionally important structural elements of the rat type IIa Na+/P-i c otransporter protein. Single point mutants with different amino acids repla ced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with subst ituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function compa rable to wild-type (WT) protein. Transport function of oocytes expressing W T protein was unchanged after exposure to the alkylating reagent 2-aminoeth yl methanethiosulfonate hydrobromide (MTSEA, 100 mu M), which indicated tha t native cysteines were inaccessible. However for one of the mutants (S460C ) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P-i transport. Alkylation in 100 mM Na+ by either cationic {[2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET ), MTSEA} or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P-i response equally well, whereas exposure to met hanethiosulfonate (MTS) reagents in 0 mM Na+ resulted in protection from th e MTS effect at depolarized potentials. This indicated that accessibility t o site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P-i suppressed the slippage current eq ually, which indicated that P-i binding could occur without cotransport. Pr e-steady state relaxations were partially suppressed and their kinetics wer e significantly faster after alkylation; nevertheless, the remaining charge movement was Na+ dependent, consistent with an intact slippage pathway. Ba sed on an alternating access model for type IIa Na+/P-i cotransport, these results suggest that site 460 is located in a region involved in conformati onal changes of the empty carrier.