title: Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.
abstract: The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that 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 methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to 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 equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based 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 conformational changes of the empty carrier.
authors.affiliation: Institute for Physiology, University of Zürich, CH-8057 Zürich, Switzerland.
authors.forename: G
authors.initials: G
authors.lastname: Lambert
authors.forename: I C
authors.initials: IC
authors.lastname: Forster
authors.forename: G
authors.initials: G
authors.lastname: Stange
authors.forename: J
authors.initials: J
authors.lastname: Biber
authors.forename: H
authors.initials: H
authors.lastname: Murer
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics*; D003545:Cysteine; D004594:Electrophysiology; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D010710:Phosphates / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary; D051381:Rats; D012694:Serine; D012964:Sodium / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D013329:Structure-Activity Relationship; D027981:Symporters; D014982:Xenopus laevis
chemical_list: D002352:Carrier Proteins; D007202:Indicators and Reagents; D010710:Phosphates; D018075:RNA, Complementary; C493556:Slc34a1 protein, rat; D050608:Sodium-Phosphate Cotransporter Proteins; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D027981:Symporters; C093171:methanethiosulfonate ethylammonium; D012694:Serine; D005020:Ethyl Methanesulfonate; D012964:Sodium; D003545:Cysteine