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Structure-function relations of the first and fourth predicted extracellular linkers of the type IIa Na+/Pi cotransporter: I. Cysteine scanning mutagenesis.

Ehnes C, Forster IC, Kohler K, Bacconi A, Stange G, Biber J, Murer H - J. Gen. Physiol. (2004)

Bottom Line: For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency.Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials.Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, Universität Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

ABSTRACT
The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693-705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

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Determination of MTS-Cys reaction rates for mutants that showed a change of activity after MTS exposure. (A) Representative recording of current from an oocyte that expressed mutant F137C after successive applications of MTSET (10 μM) (light gray bars). The cumulative exposure time (min) is indicated above each test response. Test substrates Pi (1 mM, black bars) and PFA (1 mM, dark gray bars) were applied for ∼20 s. (B and C) Pi-dependent currents at Vh = −50 mV, normalized to the initial value plotted as a function of cumulative MTS reagent exposure time for selected mutants in ECL-1 (B) and ECL-4 (C). Cells were exposed to either MTSEA (filled squares) or MTSET (open squares) for the cumulative time indicated and at the concentrations given in Table I. Continuous line is a fitted single exponential function with plateau (Eq. 1) from which the reaction rate and plateau were estimated. Each data point is pooled from ≥3 cells. Broken lines indicate plateau levels (IPi∞) estimated from the fit of Eq. 1 (Table I).
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fig4: Determination of MTS-Cys reaction rates for mutants that showed a change of activity after MTS exposure. (A) Representative recording of current from an oocyte that expressed mutant F137C after successive applications of MTSET (10 μM) (light gray bars). The cumulative exposure time (min) is indicated above each test response. Test substrates Pi (1 mM, black bars) and PFA (1 mM, dark gray bars) were applied for ∼20 s. (B and C) Pi-dependent currents at Vh = −50 mV, normalized to the initial value plotted as a function of cumulative MTS reagent exposure time for selected mutants in ECL-1 (B) and ECL-4 (C). Cells were exposed to either MTSEA (filled squares) or MTSET (open squares) for the cumulative time indicated and at the concentrations given in Table I. Continuous line is a fitted single exponential function with plateau (Eq. 1) from which the reaction rate and plateau were estimated. Each data point is pooled from ≥3 cells. Broken lines indicate plateau levels (IPi∞) estimated from the fit of Eq. 1 (Table I).

Mentions: We estimated the rate of the Cys modification reaction for mutants that showed a significant change of cotransport function after MTS exposure under standard assay conditions, by measuring IPi after successive exposures to a fixed concentration of MTS reagent. The MTS reagent concentration was chosen initially by trial and error so that a 2-min exposure resulted in an intermediate change of activity, typically in the range 30–60% of the initial response. The test concentration was found to vary over two orders of magnitude, depending on the mutation site (Table I). Fig. 4 A shows a representative record from an oocyte that expressed mutant F137C (ECL-1) during repeated exposure to 10 μM MTSET. After each exposure and washout, the response to Pi was tested and the holding current was allowed to recover to the initial baseline before the next exposure. After normalization, these data were fit with a single exponential function (Eq. 1). Under the assumption that the MTS reagent was always in excess, we could estimate the effective second order reaction rate (k*) (Table I). Impermeant (MTSET) and semi-permeant (MTSEA) reagents were used to confirm the sidedness of the reaction, i.e., to confirm that modification occurred from the external medium in the present study. Fig. 4 (B and C) illustrates the behavior of selected mutants from ECL-1 and ECL-4, respectively.


Structure-function relations of the first and fourth predicted extracellular linkers of the type IIa Na+/Pi cotransporter: I. Cysteine scanning mutagenesis.

Ehnes C, Forster IC, Kohler K, Bacconi A, Stange G, Biber J, Murer H - J. Gen. Physiol. (2004)

Determination of MTS-Cys reaction rates for mutants that showed a change of activity after MTS exposure. (A) Representative recording of current from an oocyte that expressed mutant F137C after successive applications of MTSET (10 μM) (light gray bars). The cumulative exposure time (min) is indicated above each test response. Test substrates Pi (1 mM, black bars) and PFA (1 mM, dark gray bars) were applied for ∼20 s. (B and C) Pi-dependent currents at Vh = −50 mV, normalized to the initial value plotted as a function of cumulative MTS reagent exposure time for selected mutants in ECL-1 (B) and ECL-4 (C). Cells were exposed to either MTSEA (filled squares) or MTSET (open squares) for the cumulative time indicated and at the concentrations given in Table I. Continuous line is a fitted single exponential function with plateau (Eq. 1) from which the reaction rate and plateau were estimated. Each data point is pooled from ≥3 cells. Broken lines indicate plateau levels (IPi∞) estimated from the fit of Eq. 1 (Table I).
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Related In: Results  -  Collection

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fig4: Determination of MTS-Cys reaction rates for mutants that showed a change of activity after MTS exposure. (A) Representative recording of current from an oocyte that expressed mutant F137C after successive applications of MTSET (10 μM) (light gray bars). The cumulative exposure time (min) is indicated above each test response. Test substrates Pi (1 mM, black bars) and PFA (1 mM, dark gray bars) were applied for ∼20 s. (B and C) Pi-dependent currents at Vh = −50 mV, normalized to the initial value plotted as a function of cumulative MTS reagent exposure time for selected mutants in ECL-1 (B) and ECL-4 (C). Cells were exposed to either MTSEA (filled squares) or MTSET (open squares) for the cumulative time indicated and at the concentrations given in Table I. Continuous line is a fitted single exponential function with plateau (Eq. 1) from which the reaction rate and plateau were estimated. Each data point is pooled from ≥3 cells. Broken lines indicate plateau levels (IPi∞) estimated from the fit of Eq. 1 (Table I).
Mentions: We estimated the rate of the Cys modification reaction for mutants that showed a significant change of cotransport function after MTS exposure under standard assay conditions, by measuring IPi after successive exposures to a fixed concentration of MTS reagent. The MTS reagent concentration was chosen initially by trial and error so that a 2-min exposure resulted in an intermediate change of activity, typically in the range 30–60% of the initial response. The test concentration was found to vary over two orders of magnitude, depending on the mutation site (Table I). Fig. 4 A shows a representative record from an oocyte that expressed mutant F137C (ECL-1) during repeated exposure to 10 μM MTSET. After each exposure and washout, the response to Pi was tested and the holding current was allowed to recover to the initial baseline before the next exposure. After normalization, these data were fit with a single exponential function (Eq. 1). Under the assumption that the MTS reagent was always in excess, we could estimate the effective second order reaction rate (k*) (Table I). Impermeant (MTSET) and semi-permeant (MTSEA) reagents were used to confirm the sidedness of the reaction, i.e., to confirm that modification occurred from the external medium in the present study. Fig. 4 (B and C) illustrates the behavior of selected mutants from ECL-1 and ECL-4, respectively.

Bottom Line: For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency.Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials.Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, Universität Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

ABSTRACT
The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693-705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

Show MeSH
Related in: MedlinePlus