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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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Steady state kinetics of oocytes expressing S460C. (A) Pi dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s) at 100 mM Na+. Data points are pooled from four oocytes from the same batch.  was fit to the dose–response data for each cell and the data points were normalized to the predicted maximum current. Continuous line is refit of  to the pooled data, giving a Hill coefficient, nPi = 1.04 ± 0.07 and apparent Pi affinity, KmPi = 0.081 ± 0.01 mM. (B) Na+ dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s), at 1 mM Pi. Data points are pooled from three oocytes from the same batch. Data were treated as in A. Fit of  (continuous line) gave a Hill coefficient nNa = 2.35 ± 0.21 and apparent Na+ affinity KmNa = 56.3 ± 4.0 mM. (C) Effect of PFA on the slippage mode (left) and cotransport mode (right) for WT (filled bars) (n = 9) and S460C (open bars) (n = 5). Inset shows an original recording from a cell expressing S460C: (1) response to 0.3 mM Pi, (2) response to 0.3 mM Pi and 3 mM PFA, (3) response to 3 mM PFA. Traces have been aligned to the baseline current in the absence of substrate (dashed line). For the slippage mode assay, bars represent the ratio of trace 3 response to trace 1 response. For cotransport mode assay, bars represent the ratio of trace 2 response to trace 1 response, both relative to the level in the presence of PFA alone (trace 3).
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Figure 4: Steady state kinetics of oocytes expressing S460C. (A) Pi dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s) at 100 mM Na+. Data points are pooled from four oocytes from the same batch. was fit to the dose–response data for each cell and the data points were normalized to the predicted maximum current. Continuous line is refit of to the pooled data, giving a Hill coefficient, nPi = 1.04 ± 0.07 and apparent Pi affinity, KmPi = 0.081 ± 0.01 mM. (B) Na+ dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s), at 1 mM Pi. Data points are pooled from three oocytes from the same batch. Data were treated as in A. Fit of (continuous line) gave a Hill coefficient nNa = 2.35 ± 0.21 and apparent Na+ affinity KmNa = 56.3 ± 4.0 mM. (C) Effect of PFA on the slippage mode (left) and cotransport mode (right) for WT (filled bars) (n = 9) and S460C (open bars) (n = 5). Inset shows an original recording from a cell expressing S460C: (1) response to 0.3 mM Pi, (2) response to 0.3 mM Pi and 3 mM PFA, (3) response to 3 mM PFA. Traces have been aligned to the baseline current in the absence of substrate (dashed line). For the slippage mode assay, bars represent the ratio of trace 3 response to trace 1 response. For cotransport mode assay, bars represent the ratio of trace 2 response to trace 1 response, both relative to the level in the presence of PFA alone (trace 3).

Mentions: We first confirmed that S460C exhibited a dose dependency for the respective substrates (Pi, Na+) that was consistent with the WT. These findings are shown in Fig. 4 A for the Pi-activated dose response and B for the Na+-activated dose response, pooled from representative oocytes expressing the mutant S460C. In each case, a set of original records at the substrate test concentrations is given for a representative oocyte. These were indistinguishable from the typical WT responses under the same conditions (data not shown). For both substrate activation data sets, the steady state currents at the test concentration were normalized to the maximum current predicted from a fit to the whole data set for each cell using the modified Hill equation (). The Pi-activated response was determined at 100 mM Na+ and fits to the data gave a Hill coefficient, nPi = 1.04 ± 0.1 mM and an apparent affinity for Pi (KmPi) of 0.08 ± 0.01 mM. The Na+ dose response was determined at 1 mM Pi and fits to the data gave a Hill coefficient, nNa = 2.4 ± 0 2 mM and an apparent Na+ affinity (KmNa) of 56 ± 4 mM. These parameters were sufficiently close to the previously reported values for the WT under the same measurement conditions (e.g., nPi = 0.96, KmPi = 0.057, nNa = 2.9, KmNa = 52 mM; Forster et al. 1998), as well as control oocytes expressing the WT tested in the present study (data not shown), for us to conclude that neither the apparent substrate affinities nor the inferred stoichiometry was affected by the Cys mutation. One further steady state property, which characterizes type IIa Na+/Pi cotransport, namely pH sensitivity, was also found to be unchanged in the mutant S460C when compared with the WT expressed in oocytes from the same batch. In 100 mM Na+, a reduction in superfusate pH from 7.4 to 6.2 gave a 55 ± 4% (n = 5) suppression of the Pi-induced response (1 mM total Pi) compared with 58 ± 4% (n = 5) for the WT.


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)

Steady state kinetics of oocytes expressing S460C. (A) Pi dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s) at 100 mM Na+. Data points are pooled from four oocytes from the same batch.  was fit to the dose–response data for each cell and the data points were normalized to the predicted maximum current. Continuous line is refit of  to the pooled data, giving a Hill coefficient, nPi = 1.04 ± 0.07 and apparent Pi affinity, KmPi = 0.081 ± 0.01 mM. (B) Na+ dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s), at 1 mM Pi. Data points are pooled from three oocytes from the same batch. Data were treated as in A. Fit of  (continuous line) gave a Hill coefficient nNa = 2.35 ± 0.21 and apparent Na+ affinity KmNa = 56.3 ± 4.0 mM. (C) Effect of PFA on the slippage mode (left) and cotransport mode (right) for WT (filled bars) (n = 9) and S460C (open bars) (n = 5). Inset shows an original recording from a cell expressing S460C: (1) response to 0.3 mM Pi, (2) response to 0.3 mM Pi and 3 mM PFA, (3) response to 3 mM PFA. Traces have been aligned to the baseline current in the absence of substrate (dashed line). For the slippage mode assay, bars represent the ratio of trace 3 response to trace 1 response. For cotransport mode assay, bars represent the ratio of trace 2 response to trace 1 response, both relative to the level in the presence of PFA alone (trace 3).
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Figure 4: Steady state kinetics of oocytes expressing S460C. (A) Pi dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s) at 100 mM Na+. Data points are pooled from four oocytes from the same batch. was fit to the dose–response data for each cell and the data points were normalized to the predicted maximum current. Continuous line is refit of to the pooled data, giving a Hill coefficient, nPi = 1.04 ± 0.07 and apparent Pi affinity, KmPi = 0.081 ± 0.01 mM. (B) Na+ dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s), at 1 mM Pi. Data points are pooled from three oocytes from the same batch. Data were treated as in A. Fit of (continuous line) gave a Hill coefficient nNa = 2.35 ± 0.21 and apparent Na+ affinity KmNa = 56.3 ± 4.0 mM. (C) Effect of PFA on the slippage mode (left) and cotransport mode (right) for WT (filled bars) (n = 9) and S460C (open bars) (n = 5). Inset shows an original recording from a cell expressing S460C: (1) response to 0.3 mM Pi, (2) response to 0.3 mM Pi and 3 mM PFA, (3) response to 3 mM PFA. Traces have been aligned to the baseline current in the absence of substrate (dashed line). For the slippage mode assay, bars represent the ratio of trace 3 response to trace 1 response. For cotransport mode assay, bars represent the ratio of trace 2 response to trace 1 response, both relative to the level in the presence of PFA alone (trace 3).
Mentions: We first confirmed that S460C exhibited a dose dependency for the respective substrates (Pi, Na+) that was consistent with the WT. These findings are shown in Fig. 4 A for the Pi-activated dose response and B for the Na+-activated dose response, pooled from representative oocytes expressing the mutant S460C. In each case, a set of original records at the substrate test concentrations is given for a representative oocyte. These were indistinguishable from the typical WT responses under the same conditions (data not shown). For both substrate activation data sets, the steady state currents at the test concentration were normalized to the maximum current predicted from a fit to the whole data set for each cell using the modified Hill equation (). The Pi-activated response was determined at 100 mM Na+ and fits to the data gave a Hill coefficient, nPi = 1.04 ± 0.1 mM and an apparent affinity for Pi (KmPi) of 0.08 ± 0.01 mM. The Na+ dose response was determined at 1 mM Pi and fits to the data gave a Hill coefficient, nNa = 2.4 ± 0 2 mM and an apparent Na+ affinity (KmNa) of 56 ± 4 mM. These parameters were sufficiently close to the previously reported values for the WT under the same measurement conditions (e.g., nPi = 0.96, KmPi = 0.057, nNa = 2.9, KmNa = 52 mM; Forster et al. 1998), as well as control oocytes expressing the WT tested in the present study (data not shown), for us to conclude that neither the apparent substrate affinities nor the inferred stoichiometry was affected by the Cys mutation. One further steady state property, which characterizes type IIa Na+/Pi cotransport, namely pH sensitivity, was also found to be unchanged in the mutant S460C when compared with the WT expressed in oocytes from the same batch. In 100 mM Na+, a reduction in superfusate pH from 7.4 to 6.2 gave a 55 ± 4% (n = 5) suppression of the Pi-induced response (1 mM total Pi) compared with 58 ± 4% (n = 5) for the WT.

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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Related in: MedlinePlus