Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of alkylation by MTSEA on Pi and PFA response for cells expressing S460C. Substrate was applied during the period indicated by the bar. Cell was voltage clamped to −50 mV. Note that after alkylation, the 3 mM Pi and 3 mM PFA responses superimpose and are identical to the PFA response before alkylation. Vh = −50 mV.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230544&req=5

Figure 6: Effect of alkylation by MTSEA on Pi and PFA response for cells expressing S460C. Substrate was applied during the period indicated by the bar. Cell was voltage clamped to −50 mV. Note that after alkylation, the 3 mM Pi and 3 mM PFA responses superimpose and are identical to the PFA response before alkylation. Vh = −50 mV.

Mentions: A noteworthy result of incubation in MTSEA on the Pi response was the reduction of holding current during Pi application (Fig. 2 A). We investigated this further by testing the response to 3 mM Pi or 3 mM PFA before and after 100 μM MTSEA incubation as shown for a representative oocyte in Fig. 6. These substrate concentrations were chosen to ensure saturation of the responses. After alkylation, the response to PFA remained unchanged, whereas the Pi response was now identical to the PFA response. Moreover, this behavior was found to be consistent for all potentials in the range −80 to 0 mV (data not shown). This result suggested that: (a) alkylation of Cys-460 did not affect the slippage mode, (b) Na+, the cation responsible for slippage current, was still able to bind to the carrier after alkylation, and (c) Pi can still bind to the carrier after alkylation, but subsequent cotransport was suppressed. Noninjected oocytes from the same batch showed small changes in holding current with either Pi or PFA application, under the same conditions, but which were <10% of the responses recorded from S460C-expressing oocytes.


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)

Effect of alkylation by MTSEA on Pi and PFA response for cells expressing S460C. Substrate was applied during the period indicated by the bar. Cell was voltage clamped to −50 mV. Note that after alkylation, the 3 mM Pi and 3 mM PFA responses superimpose and are identical to the PFA response before alkylation. Vh = −50 mV.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Effect of alkylation by MTSEA on Pi and PFA response for cells expressing S460C. Substrate was applied during the period indicated by the bar. Cell was voltage clamped to −50 mV. Note that after alkylation, the 3 mM Pi and 3 mM PFA responses superimpose and are identical to the PFA response before alkylation. Vh = −50 mV.
Mentions: A noteworthy result of incubation in MTSEA on the Pi response was the reduction of holding current during Pi application (Fig. 2 A). We investigated this further by testing the response to 3 mM Pi or 3 mM PFA before and after 100 μM MTSEA incubation as shown for a representative oocyte in Fig. 6. These substrate concentrations were chosen to ensure saturation of the responses. After alkylation, the response to PFA remained unchanged, whereas the Pi response was now identical to the PFA response. Moreover, this behavior was found to be consistent for all potentials in the range −80 to 0 mV (data not shown). This result suggested that: (a) alkylation of Cys-460 did not affect the slippage mode, (b) Na+, the cation responsible for slippage current, was still able to bind to the carrier after alkylation, and (c) Pi can still bind to the carrier after alkylation, but subsequent cotransport was suppressed. Noninjected oocytes from the same batch showed small changes in holding current with either Pi or PFA application, under the same conditions, but which were <10% of the responses recorded from S460C-expressing oocytes.

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