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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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Membrane potential protects against MTSEA suppression of Pi response only in the absence of external Na+. (A) Original recordings from two representative oocytes (top and bottom) from the same donor frog expressing S460C before and after a 2-min exposure to 10 μM MTSEA at different holding potentials. After alkylation, the response was tested each time with 1 mM Pi at −50 mV in ND100. (Top, 1) Initial response, (2) response after alkylation at +20 mV holding potential in ND100. (Bottom, 1) Initial response, (2) response after alkylation at +20 mV in ND0, (3) response after alkylation at −50 mV in ND0, (4) response after alkylation at −50 mV in ND100. The dashed line represents the initial holding current level before Pi application. (B) Pooled data of the inhibition of the Pi response after alkylation in 0 mM Na+, at three holding potentials. n = 3 (−20 mV, 0 mV); n = 5 (+20 mV). For all cells, after reexposure to MTSEA at −50 mV in ND100 solution, the Pi response was the same as the PFA response. The percent change in the Pi-induced electrogenic response was expressed as: 100 · [1 − (Ip+ + Is)/(Ip− + Is)], where Ip− and Ip+ are the Pi-induced current before and after MTSEA exposure, respectively, Is is the PFA-induced change in holding current (slippage current), with all currents expressed as magnitudes. It was assumed that the slippage was fully suppressed by 3 mM PFA so that the true Pi-induced response for saturating Pi was given by the change in current relative to the holding current during PFA exposure.
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Figure 8: Membrane potential protects against MTSEA suppression of Pi response only in the absence of external Na+. (A) Original recordings from two representative oocytes (top and bottom) from the same donor frog expressing S460C before and after a 2-min exposure to 10 μM MTSEA at different holding potentials. After alkylation, the response was tested each time with 1 mM Pi at −50 mV in ND100. (Top, 1) Initial response, (2) response after alkylation at +20 mV holding potential in ND100. (Bottom, 1) Initial response, (2) response after alkylation at +20 mV in ND0, (3) response after alkylation at −50 mV in ND0, (4) response after alkylation at −50 mV in ND100. The dashed line represents the initial holding current level before Pi application. (B) Pooled data of the inhibition of the Pi response after alkylation in 0 mM Na+, at three holding potentials. n = 3 (−20 mV, 0 mV); n = 5 (+20 mV). For all cells, after reexposure to MTSEA at −50 mV in ND100 solution, the Pi response was the same as the PFA response. The percent change in the Pi-induced electrogenic response was expressed as: 100 · [1 − (Ip+ + Is)/(Ip− + Is)], where Ip− and Ip+ are the Pi-induced current before and after MTSEA exposure, respectively, Is is the PFA-induced change in holding current (slippage current), with all currents expressed as magnitudes. It was assumed that the slippage was fully suppressed by 3 mM PFA so that the true Pi-induced response for saturating Pi was given by the change in current relative to the holding current during PFA exposure.

Mentions: In a final set of experiments, we investigated whether holding potential (Vh) during MTSEA application would also protect against alkylation as reported in the case of SGLT1 (Loo et al. 1998). As illustrated by the original recordings from representative oocytes expressing S460C (Fig. 8 A), when MTSEA was applied during a depolarization to +20 mV in ND100 solution, the subsequent response to Pi at −50 mV was identical to that obtained when MTSEA was applied at −50 mV (Fig. 6). This indicated that in ND100 depolarization to +20 mV did not protect Cys-460. However, when MTSEA was applied in ND0 solution, the magnitude of the Pi response was now dependent on Vh, whereby less inhibition was observed at Vh = +20 mV compared with the response after alkylation at −50 mV. We repeated this protocol for individual oocytes voltage clamped to Vh = +20, 0, and −20 mV during MTSEA exposure, and the pooled results (Fig. 8 B) indicate a clear voltage dependence of inhibition of response by the MTS reagent in 0 mM Na+. For each oocyte tested, we confirmed that exposure to MTSEA at Vh = −50 mV in 100 mM Na+ gave complete inhibition (i.e., the same response during PFA exposure; Fig. 6). We were unable to determine whether at more depolarized Vh further protection of the MTS action was possible because continuous voltage clamping of oocytes at potentials exceeding +20 mV during the MTSEA application period resulted in an irreversible and progressive increase in endogenous leak current. This protocol was also repeated with the anionic MTSES with similar results (data not shown). This confirmed that accessibility to Cys-460 was independent of the charge of the alkylating reagent.


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)

Membrane potential protects against MTSEA suppression of Pi response only in the absence of external Na+. (A) Original recordings from two representative oocytes (top and bottom) from the same donor frog expressing S460C before and after a 2-min exposure to 10 μM MTSEA at different holding potentials. After alkylation, the response was tested each time with 1 mM Pi at −50 mV in ND100. (Top, 1) Initial response, (2) response after alkylation at +20 mV holding potential in ND100. (Bottom, 1) Initial response, (2) response after alkylation at +20 mV in ND0, (3) response after alkylation at −50 mV in ND0, (4) response after alkylation at −50 mV in ND100. The dashed line represents the initial holding current level before Pi application. (B) Pooled data of the inhibition of the Pi response after alkylation in 0 mM Na+, at three holding potentials. n = 3 (−20 mV, 0 mV); n = 5 (+20 mV). For all cells, after reexposure to MTSEA at −50 mV in ND100 solution, the Pi response was the same as the PFA response. The percent change in the Pi-induced electrogenic response was expressed as: 100 · [1 − (Ip+ + Is)/(Ip− + Is)], where Ip− and Ip+ are the Pi-induced current before and after MTSEA exposure, respectively, Is is the PFA-induced change in holding current (slippage current), with all currents expressed as magnitudes. It was assumed that the slippage was fully suppressed by 3 mM PFA so that the true Pi-induced response for saturating Pi was given by the change in current relative to the holding current during PFA exposure.
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Figure 8: Membrane potential protects against MTSEA suppression of Pi response only in the absence of external Na+. (A) Original recordings from two representative oocytes (top and bottom) from the same donor frog expressing S460C before and after a 2-min exposure to 10 μM MTSEA at different holding potentials. After alkylation, the response was tested each time with 1 mM Pi at −50 mV in ND100. (Top, 1) Initial response, (2) response after alkylation at +20 mV holding potential in ND100. (Bottom, 1) Initial response, (2) response after alkylation at +20 mV in ND0, (3) response after alkylation at −50 mV in ND0, (4) response after alkylation at −50 mV in ND100. The dashed line represents the initial holding current level before Pi application. (B) Pooled data of the inhibition of the Pi response after alkylation in 0 mM Na+, at three holding potentials. n = 3 (−20 mV, 0 mV); n = 5 (+20 mV). For all cells, after reexposure to MTSEA at −50 mV in ND100 solution, the Pi response was the same as the PFA response. The percent change in the Pi-induced electrogenic response was expressed as: 100 · [1 − (Ip+ + Is)/(Ip− + Is)], where Ip− and Ip+ are the Pi-induced current before and after MTSEA exposure, respectively, Is is the PFA-induced change in holding current (slippage current), with all currents expressed as magnitudes. It was assumed that the slippage was fully suppressed by 3 mM PFA so that the true Pi-induced response for saturating Pi was given by the change in current relative to the holding current during PFA exposure.
Mentions: In a final set of experiments, we investigated whether holding potential (Vh) during MTSEA application would also protect against alkylation as reported in the case of SGLT1 (Loo et al. 1998). As illustrated by the original recordings from representative oocytes expressing S460C (Fig. 8 A), when MTSEA was applied during a depolarization to +20 mV in ND100 solution, the subsequent response to Pi at −50 mV was identical to that obtained when MTSEA was applied at −50 mV (Fig. 6). This indicated that in ND100 depolarization to +20 mV did not protect Cys-460. However, when MTSEA was applied in ND0 solution, the magnitude of the Pi response was now dependent on Vh, whereby less inhibition was observed at Vh = +20 mV compared with the response after alkylation at −50 mV. We repeated this protocol for individual oocytes voltage clamped to Vh = +20, 0, and −20 mV during MTSEA exposure, and the pooled results (Fig. 8 B) indicate a clear voltage dependence of inhibition of response by the MTS reagent in 0 mM Na+. For each oocyte tested, we confirmed that exposure to MTSEA at Vh = −50 mV in 100 mM Na+ gave complete inhibition (i.e., the same response during PFA exposure; Fig. 6). We were unable to determine whether at more depolarized Vh further protection of the MTS action was possible because continuous voltage clamping of oocytes at potentials exceeding +20 mV during the MTSEA application period resulted in an irreversible and progressive increase in endogenous leak current. This protocol was also repeated with the anionic MTSES with similar results (data not shown). This confirmed that accessibility to Cys-460 was independent of the charge of the alkylating reagent.

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