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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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Alkylation using MTSEA leads to a suppression of the electrogenic response in oocytes expressing mutant S460C. (A) Comparison of the Pi-induced current for an oocyte expressing the WT NaPi-IIa before and after exposure to 100 μM MTSEA (left) and two oocytes expressing S460C before and after exposure to 100 μM MTSEA or MTSET for 2 min (right). Bars represent time of application of 1 mM Pi. Dashed line indicates baseline holding current level. Note that Pi induces an upward deflection in the holding current after alkylation. (B) Restoration of Pi response by 15-min incubation in 10 mM DTT (right) for a cell expressing S460C. Incubation in 1.5 μM MTSEA exposure (middle) inhibited the initial Pi response (left) by 80%. (C) Dependency of the Pi response on MTSEA concentration. Inset gives a set of original records showing Pi response (applied during bar) for an oocyte expressing S460C after exposure to successive 10-fold increasing doses of MTSEA from 0.001 to 10 μM. Scale: vertical 50 nA, horizontal 20 s. For this cell, after exposure at 10 μM MTSEA, Pi induced an upward deflection of the baseline current. There was no change in the response after exposure to 100 μM MTSEA. Dose response is pooled from five oocytes. The Pi-induced response was calculated relative to the Pi-induced current after alkylation in 100 μM MTSEA, and was then normalized to the initial response for each cell. Curve is a fit of  to the data, which gave an apparent half-maximal concentration = 0.5 μM.
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Figure 2: Alkylation using MTSEA leads to a suppression of the electrogenic response in oocytes expressing mutant S460C. (A) Comparison of the Pi-induced current for an oocyte expressing the WT NaPi-IIa before and after exposure to 100 μM MTSEA (left) and two oocytes expressing S460C before and after exposure to 100 μM MTSEA or MTSET for 2 min (right). Bars represent time of application of 1 mM Pi. Dashed line indicates baseline holding current level. Note that Pi induces an upward deflection in the holding current after alkylation. (B) Restoration of Pi response by 15-min incubation in 10 mM DTT (right) for a cell expressing S460C. Incubation in 1.5 μM MTSEA exposure (middle) inhibited the initial Pi response (left) by 80%. (C) Dependency of the Pi response on MTSEA concentration. Inset gives a set of original records showing Pi response (applied during bar) for an oocyte expressing S460C after exposure to successive 10-fold increasing doses of MTSEA from 0.001 to 10 μM. Scale: vertical 50 nA, horizontal 20 s. For this cell, after exposure at 10 μM MTSEA, Pi induced an upward deflection of the baseline current. There was no change in the response after exposure to 100 μM MTSEA. Dose response is pooled from five oocytes. The Pi-induced response was calculated relative to the Pi-induced current after alkylation in 100 μM MTSEA, and was then normalized to the initial response for each cell. Curve is a fit of to the data, which gave an apparent half-maximal concentration = 0.5 μM.

Mentions: To test if alkylation of the cysteine residues by the methanethiosulfonate derivative MTSEA would affect the basic transport function, as indicated by the above electrophysiological assay, we incubated oocytes expressing these six active mutants, as well as the WT protein, in 100 μM MTSEA, and then retested for activity under the same conditions as before. The Pi-induced change in holding current, exhibited by the WT (Fig. 2 A) as well as five of the active mutants (S318C, S373C, A393C, S532C, and S538C; data not shown), was unaffected by MTSEA (Table ). In contrast, after incubation in MTSEA, the electrogenic response of mutant S460C showed a significant inhibition (Fig. 2 A) during application of 1 mM Pi. Moreover, prolonged incubation (up to 30 min) in the standard bath medium did not lead to a restoration of function (data not shown). We also found that after alkylation, 32P uptake of S460C was completely suppressed (data not shown), which confirmed that Pi transport was fully inhibited. To demonstrate that the suppression of electrogenic response was an effect of the alkylation and not simply due to the addition of charge in this region (MTSEA is positively charged), we repeated the experiment with the negatively charged MTS reagent, MTSES. Like MTSEA, incubation in 100 μM MTSES, also induced a positive shift in the baseline current during Pi application; i.e., the normal inward current induced by Pi was fully suppressed (data not shown). We also incubated oocytes in 100 μM MTSET, which has been reported to be less permeant than MTSEA (Holmgren et al. 1996) and we obtained the same suppression of Pi response (n = 3). A representative record is shown in Fig. 2 A. This result suggested that both MTSEA and MTSET were acting extracellularly. Moreover, even at high concentrations (1 mM), all three reagents (MTSEA, MTSET, MTSES) had no effect on the electrogenic WT response (data not shown). This was also confirmed in uptake experiments in which there was no statistical difference in 32P uptake between WT-expressing oocytes exposed to 1 mM MTSEA, MTSET, or MTSES and control oocytes. In each case, the 32P uptake was >20-fold higher than that of water-injected oocytes from the same donor frog (results not shown). In contrast to the lack of effect of MTSEA, MTSET, and MTSES on the WT, we did observe a dose dependency of 32P uptake in WT-expressing oocytes exposed to the membrane-permeant reagent methyl-MTS (Lambert, G., J. Biber, and H. Murer, manuscript submitted for publication). This further supported our conclusion that MTSEA, MTSES, and MTSET were only acting extracellularly. All remaining experiments were performed with MTSEA unless otherwise indicated.


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)

Alkylation using MTSEA leads to a suppression of the electrogenic response in oocytes expressing mutant S460C. (A) Comparison of the Pi-induced current for an oocyte expressing the WT NaPi-IIa before and after exposure to 100 μM MTSEA (left) and two oocytes expressing S460C before and after exposure to 100 μM MTSEA or MTSET for 2 min (right). Bars represent time of application of 1 mM Pi. Dashed line indicates baseline holding current level. Note that Pi induces an upward deflection in the holding current after alkylation. (B) Restoration of Pi response by 15-min incubation in 10 mM DTT (right) for a cell expressing S460C. Incubation in 1.5 μM MTSEA exposure (middle) inhibited the initial Pi response (left) by 80%. (C) Dependency of the Pi response on MTSEA concentration. Inset gives a set of original records showing Pi response (applied during bar) for an oocyte expressing S460C after exposure to successive 10-fold increasing doses of MTSEA from 0.001 to 10 μM. Scale: vertical 50 nA, horizontal 20 s. For this cell, after exposure at 10 μM MTSEA, Pi induced an upward deflection of the baseline current. There was no change in the response after exposure to 100 μM MTSEA. Dose response is pooled from five oocytes. The Pi-induced response was calculated relative to the Pi-induced current after alkylation in 100 μM MTSEA, and was then normalized to the initial response for each cell. Curve is a fit of  to the data, which gave an apparent half-maximal concentration = 0.5 μM.
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Related In: Results  -  Collection

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Figure 2: Alkylation using MTSEA leads to a suppression of the electrogenic response in oocytes expressing mutant S460C. (A) Comparison of the Pi-induced current for an oocyte expressing the WT NaPi-IIa before and after exposure to 100 μM MTSEA (left) and two oocytes expressing S460C before and after exposure to 100 μM MTSEA or MTSET for 2 min (right). Bars represent time of application of 1 mM Pi. Dashed line indicates baseline holding current level. Note that Pi induces an upward deflection in the holding current after alkylation. (B) Restoration of Pi response by 15-min incubation in 10 mM DTT (right) for a cell expressing S460C. Incubation in 1.5 μM MTSEA exposure (middle) inhibited the initial Pi response (left) by 80%. (C) Dependency of the Pi response on MTSEA concentration. Inset gives a set of original records showing Pi response (applied during bar) for an oocyte expressing S460C after exposure to successive 10-fold increasing doses of MTSEA from 0.001 to 10 μM. Scale: vertical 50 nA, horizontal 20 s. For this cell, after exposure at 10 μM MTSEA, Pi induced an upward deflection of the baseline current. There was no change in the response after exposure to 100 μM MTSEA. Dose response is pooled from five oocytes. The Pi-induced response was calculated relative to the Pi-induced current after alkylation in 100 μM MTSEA, and was then normalized to the initial response for each cell. Curve is a fit of to the data, which gave an apparent half-maximal concentration = 0.5 μM.
Mentions: To test if alkylation of the cysteine residues by the methanethiosulfonate derivative MTSEA would affect the basic transport function, as indicated by the above electrophysiological assay, we incubated oocytes expressing these six active mutants, as well as the WT protein, in 100 μM MTSEA, and then retested for activity under the same conditions as before. The Pi-induced change in holding current, exhibited by the WT (Fig. 2 A) as well as five of the active mutants (S318C, S373C, A393C, S532C, and S538C; data not shown), was unaffected by MTSEA (Table ). In contrast, after incubation in MTSEA, the electrogenic response of mutant S460C showed a significant inhibition (Fig. 2 A) during application of 1 mM Pi. Moreover, prolonged incubation (up to 30 min) in the standard bath medium did not lead to a restoration of function (data not shown). We also found that after alkylation, 32P uptake of S460C was completely suppressed (data not shown), which confirmed that Pi transport was fully inhibited. To demonstrate that the suppression of electrogenic response was an effect of the alkylation and not simply due to the addition of charge in this region (MTSEA is positively charged), we repeated the experiment with the negatively charged MTS reagent, MTSES. Like MTSEA, incubation in 100 μM MTSES, also induced a positive shift in the baseline current during Pi application; i.e., the normal inward current induced by Pi was fully suppressed (data not shown). We also incubated oocytes in 100 μM MTSET, which has been reported to be less permeant than MTSEA (Holmgren et al. 1996) and we obtained the same suppression of Pi response (n = 3). A representative record is shown in Fig. 2 A. This result suggested that both MTSEA and MTSET were acting extracellularly. Moreover, even at high concentrations (1 mM), all three reagents (MTSEA, MTSET, MTSES) had no effect on the electrogenic WT response (data not shown). This was also confirmed in uptake experiments in which there was no statistical difference in 32P uptake between WT-expressing oocytes exposed to 1 mM MTSEA, MTSET, or MTSES and control oocytes. In each case, the 32P uptake was >20-fold higher than that of water-injected oocytes from the same donor frog (results not shown). In contrast to the lack of effect of MTSEA, MTSET, and MTSES on the WT, we did observe a dose dependency of 32P uptake in WT-expressing oocytes exposed to the membrane-permeant reagent methyl-MTS (Lambert, G., J. Biber, and H. Murer, manuscript submitted for publication). This further supported our conclusion that MTSEA, MTSES, and MTSET were only acting extracellularly. All remaining experiments were performed with MTSEA unless otherwise indicated.

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