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Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

Lambert G, Forster IC, Stange G, Biber J, Murer H - J. Gen. Physiol. (1999)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physiology, University of Zürich, CH-8057 Zürich, Switzerland.

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.

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Topological representation of the rat type IIa sodium phosphate cotransporter (rat NaPi-IIa). Scheme is based on current hydropathy data and a recent topology study (Lambert et al. 1999) in which eight membrane spanning regions (TM1–TM8) are predicted with extracellular loops between TM1–TM2, TM3–TM4, TM5–TM6, and TM7–TM8, and both the carboxy and amino termini are intracellular. (•) Positions of 15 residues that were individually mutated to cysteines (see methods and materials).
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Figure 1: Topological representation of the rat type IIa sodium phosphate cotransporter (rat NaPi-IIa). Scheme is based on current hydropathy data and a recent topology study (Lambert et al. 1999) in which eight membrane spanning regions (TM1–TM8) are predicted with extracellular loops between TM1–TM2, TM3–TM4, TM5–TM6, and TM7–TM8, and both the carboxy and amino termini are intracellular. (•) Positions of 15 residues that were individually mutated to cysteines (see methods and materials).

Mentions: In the present study, we have adopted a cysteine replacement strategy and substituted 15 selected amino acids with cysteine residues with the aim of identifying sites where the reaction with MTS reagents would lead to a detectable change in transport function. Having no precedent for the selection of residues, we based our choice on the following criteria: (a) residues located between hydrophobic and hydrophilic regions were chosen because these intervening regions could be likely candidates for substrate binding or conformational changes during the transport process (Lo and Silverman 1998a; Loo et al. 1998) (see Fig. 1), (b) serine or alanine residues, where present, were selected for cysteine substitution to minimize changes in the protein, and (c) residues in the amino or carboxy termini were not mutated because these are most likely located intracellularly. Furthermore, based on the evolutionary tree of Na+-coupled Pi cotransporters, both termini are located in quite variable regions (Biber et al. 1996, Biber et al. 1998; Murer and Biber 1997), which suggested they are not involved in basic substrate binding or transport processes.


Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

Lambert G, Forster IC, Stange G, Biber J, Murer H - J. Gen. Physiol. (1999)

Topological representation of the rat type IIa sodium phosphate cotransporter (rat NaPi-IIa). Scheme is based on current hydropathy data and a recent topology study (Lambert et al. 1999) in which eight membrane spanning regions (TM1–TM8) are predicted with extracellular loops between TM1–TM2, TM3–TM4, TM5–TM6, and TM7–TM8, and both the carboxy and amino termini are intracellular. (•) Positions of 15 residues that were individually mutated to cysteines (see methods and materials).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230544&req=5

Figure 1: Topological representation of the rat type IIa sodium phosphate cotransporter (rat NaPi-IIa). Scheme is based on current hydropathy data and a recent topology study (Lambert et al. 1999) in which eight membrane spanning regions (TM1–TM8) are predicted with extracellular loops between TM1–TM2, TM3–TM4, TM5–TM6, and TM7–TM8, and both the carboxy and amino termini are intracellular. (•) Positions of 15 residues that were individually mutated to cysteines (see methods and materials).
Mentions: In the present study, we have adopted a cysteine replacement strategy and substituted 15 selected amino acids with cysteine residues with the aim of identifying sites where the reaction with MTS reagents would lead to a detectable change in transport function. Having no precedent for the selection of residues, we based our choice on the following criteria: (a) residues located between hydrophobic and hydrophilic regions were chosen because these intervening regions could be likely candidates for substrate binding or conformational changes during the transport process (Lo and Silverman 1998a; Loo et al. 1998) (see Fig. 1), (b) serine or alanine residues, where present, were selected for cysteine substitution to minimize changes in the protein, and (c) residues in the amino or carboxy termini were not mutated because these are most likely located intracellularly. Furthermore, based on the evolutionary tree of Na+-coupled Pi cotransporters, both termini are located in quite variable regions (Biber et al. 1996, Biber et al. 1998; Murer and Biber 1997), which suggested they are not involved in basic substrate binding or transport processes.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physiology, University of Zürich, CH-8057 Zürich, Switzerland.

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.

Show MeSH
Related in: MedlinePlus