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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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Immunodetection of WT and S460C protein. (A) Western blot obtained from a pool of five oocytes injected either with water, WT, or S460C cRNA. 10 μl of the lysates was separated on a 9% SDS gel and, after blotting, immunoreactive proteins were visualized by incubation with an antibody against the rat NaPi IIa NH2 terminus. This blot confirms that lysate from oocytes expressing S460C presents a similar band (97 kD) to the WT. (B) Streptavidin precipitation of oocyte lysate obtained from pools of five oocytes injected either with water, wild type, or S460C cRNA and incubated with 100 μM MTSEA-Biotin. Cells were then homogenized in 100 μl buffer (see materials and methods) and 90 μl of each lysate was incubated with Streptavidin beads. After washing, bound proteins were eluted with loading buffer (see materials and methods) and the elute was then treated as for A. Single band at 97 kD confirms that the alkylated protein was S460C.
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Figure 3: Immunodetection of WT and S460C protein. (A) Western blot obtained from a pool of five oocytes injected either with water, WT, or S460C cRNA. 10 μl of the lysates was separated on a 9% SDS gel and, after blotting, immunoreactive proteins were visualized by incubation with an antibody against the rat NaPi IIa NH2 terminus. This blot confirms that lysate from oocytes expressing S460C presents a similar band (97 kD) to the WT. (B) Streptavidin precipitation of oocyte lysate obtained from pools of five oocytes injected either with water, wild type, or S460C cRNA and incubated with 100 μM MTSEA-Biotin. Cells were then homogenized in 100 μl buffer (see materials and methods) and 90 μl of each lysate was incubated with Streptavidin beads. After washing, bound proteins were eluted with loading buffer (see materials and methods) and the elute was then treated as for A. Single band at 97 kD confirms that the alkylated protein was S460C.

Mentions: Finally, to establish that the loss of transport function by S460C was due to a specific reaction of MTSEA with the Cys-460, we incubated oocytes expressing mutant S460C, as well as the WT protein, in biotin-labeled MTSEA (biotin-MTSEA) and precipitated the protein with immobilized streptavidin (see materials and methods). Expression of both proteins was confirmed by Western blot of the lysate before streptavidin precipitation. This showed that both were expressed at comparable levels (Fig. 3 A). However, as indicated by the immunoprecipitation shown in Fig. 3 B, only the mutant protein and not the WT could be precipitated after incubation with the biotin labeled MTSEA.


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)

Immunodetection of WT and S460C protein. (A) Western blot obtained from a pool of five oocytes injected either with water, WT, or S460C cRNA. 10 μl of the lysates was separated on a 9% SDS gel and, after blotting, immunoreactive proteins were visualized by incubation with an antibody against the rat NaPi IIa NH2 terminus. This blot confirms that lysate from oocytes expressing S460C presents a similar band (97 kD) to the WT. (B) Streptavidin precipitation of oocyte lysate obtained from pools of five oocytes injected either with water, wild type, or S460C cRNA and incubated with 100 μM MTSEA-Biotin. Cells were then homogenized in 100 μl buffer (see materials and methods) and 90 μl of each lysate was incubated with Streptavidin beads. After washing, bound proteins were eluted with loading buffer (see materials and methods) and the elute was then treated as for A. Single band at 97 kD confirms that the alkylated protein was S460C.
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Related In: Results  -  Collection

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Figure 3: Immunodetection of WT and S460C protein. (A) Western blot obtained from a pool of five oocytes injected either with water, WT, or S460C cRNA. 10 μl of the lysates was separated on a 9% SDS gel and, after blotting, immunoreactive proteins were visualized by incubation with an antibody against the rat NaPi IIa NH2 terminus. This blot confirms that lysate from oocytes expressing S460C presents a similar band (97 kD) to the WT. (B) Streptavidin precipitation of oocyte lysate obtained from pools of five oocytes injected either with water, wild type, or S460C cRNA and incubated with 100 μM MTSEA-Biotin. Cells were then homogenized in 100 μl buffer (see materials and methods) and 90 μl of each lysate was incubated with Streptavidin beads. After washing, bound proteins were eluted with loading buffer (see materials and methods) and the elute was then treated as for A. Single band at 97 kD confirms that the alkylated protein was S460C.
Mentions: Finally, to establish that the loss of transport function by S460C was due to a specific reaction of MTSEA with the Cys-460, we incubated oocytes expressing mutant S460C, as well as the WT protein, in biotin-labeled MTSEA (biotin-MTSEA) and precipitated the protein with immobilized streptavidin (see materials and methods). Expression of both proteins was confirmed by Western blot of the lysate before streptavidin precipitation. This showed that both were expressed at comparable levels (Fig. 3 A). However, as indicated by the immunoprecipitation shown in Fig. 3 B, only the mutant protein and not the WT could be precipitated after incubation with the biotin labeled MTSEA.

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