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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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Simulations of slippage mode behavior. (A) Four state kinetic scheme used to simulate the slippage mode. (B) Simulated currents in response to voltage steps indicated in the inset. The parameters for the simulations on the left were: k012 = 8, 000 M−1 s−1, k021 = 2,000 s−1, k023 = 5 s−1, k014 = 120 s−1, k041 = 60 s−1, k034 = 100 s−1, k043 = 1,000 M−1 s−1, α′ = 0.3, α″ = 0.3, δ = 0.4, Nao = 0.1 M, Nai = 0.01 M, T = 20°C. The effect of alkylation (right) was simulated by a 10-fold increase in k014 and k041. (C) Expanded view of the traces in A, which also shows the steady state current levels under the two conditions. The broken line indicates zero baseline current. Note that the current scale (eu, electronic units) gives the apparent charge movement per cotransporter.
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FA1: Simulations of slippage mode behavior. (A) Four state kinetic scheme used to simulate the slippage mode. (B) Simulated currents in response to voltage steps indicated in the inset. The parameters for the simulations on the left were: k012 = 8, 000 M−1 s−1, k021 = 2,000 s−1, k023 = 5 s−1, k014 = 120 s−1, k041 = 60 s−1, k034 = 100 s−1, k043 = 1,000 M−1 s−1, α′ = 0.3, α″ = 0.3, δ = 0.4, Nao = 0.1 M, Nai = 0.01 M, T = 20°C. The effect of alkylation (right) was simulated by a 10-fold increase in k014 and k041. (C) Expanded view of the traces in A, which also shows the steady state current levels under the two conditions. The broken line indicates zero baseline current. Note that the current scale (eu, electronic units) gives the apparent charge movement per cotransporter.

Mentions: The slippage mode was modeled as the four-state “iso uni uni” system depicted in 1 A (Segel 1975) and corresponding to states 1, 2, 9, and 10 of the full model in 9. Transitions between states were modeled according to the Eyring–Boltzmann transition rate theory (Adrian 1978). We assumed that the empty carrier has a valency, zc = −1, that moves an electrical distance δ through the membrane between the external and internal Na+ binding sites. A single Na+ ion moves an equivalent electrical distance α′ on the cis side and α″ on the trans side to the respective binding sites (Läuger 1991; Parent et al. 1992) so that α′ + δ + α″ = 1. The translocation (step 2 ⇔ 3) with Na+ bound is electroneutral. The eight pseudo first order rate constants, assuming symmetrical energy barriers for each transition, are then given by: \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\begin{matrix}k_{12}={\mathrm{Na}}_{{\mathrm{0}}}k^{0}_{12}{\mathrm{exp}} \left \left(-a{\mathrm{^{\prime}{\mu}}}\right) \right \\ k_{21}=k^{0}_{21}{\mathrm{exp}} \left \left(a{\mathrm{^{\prime}{\mu}}}\right) \right \\ k_{23}=k^{0}_{23}\\ k_{32}=k^{0}_{32}\\ k_{34}=k^{0}_{34}{\mathrm{exp}} \left \left(-a{\mathrm{^{\prime\prime}{\mu}}}\right) \right \\ k_{43}={\mathrm{Na}}_{{\mathrm{i}}}k^{0}_{43}{\mathrm{exp}} \left \left(a{\mathrm{^{\prime\prime}{\mu}}}\right) \right \\ k_{14}=k^{0}_{14}{\mathrm{exp}} \left \left({\mathrm{{\delta}{\mu}}}\right) \right \\ k_{41}=k^{0}_{41}{\mathrm{exp}} \left \left(-{\mathrm{{\delta}{\mu}}}\right) \right \end{matrix}\end{equation*}\end{document} and μ = eV/2kT, where, Nao and Nai are the external and internal Na+ concentrations (M), respectively, V is the transmembrane voltage, e is the electronic charge, k is Boltzmann's constant, T is the absolute temperature, and k012, k021, etc., are the rate constants at V = 0.


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)

Simulations of slippage mode behavior. (A) Four state kinetic scheme used to simulate the slippage mode. (B) Simulated currents in response to voltage steps indicated in the inset. The parameters for the simulations on the left were: k012 = 8, 000 M−1 s−1, k021 = 2,000 s−1, k023 = 5 s−1, k014 = 120 s−1, k041 = 60 s−1, k034 = 100 s−1, k043 = 1,000 M−1 s−1, α′ = 0.3, α″ = 0.3, δ = 0.4, Nao = 0.1 M, Nai = 0.01 M, T = 20°C. The effect of alkylation (right) was simulated by a 10-fold increase in k014 and k041. (C) Expanded view of the traces in A, which also shows the steady state current levels under the two conditions. The broken line indicates zero baseline current. Note that the current scale (eu, electronic units) gives the apparent charge movement per cotransporter.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230544&req=5

FA1: Simulations of slippage mode behavior. (A) Four state kinetic scheme used to simulate the slippage mode. (B) Simulated currents in response to voltage steps indicated in the inset. The parameters for the simulations on the left were: k012 = 8, 000 M−1 s−1, k021 = 2,000 s−1, k023 = 5 s−1, k014 = 120 s−1, k041 = 60 s−1, k034 = 100 s−1, k043 = 1,000 M−1 s−1, α′ = 0.3, α″ = 0.3, δ = 0.4, Nao = 0.1 M, Nai = 0.01 M, T = 20°C. The effect of alkylation (right) was simulated by a 10-fold increase in k014 and k041. (C) Expanded view of the traces in A, which also shows the steady state current levels under the two conditions. The broken line indicates zero baseline current. Note that the current scale (eu, electronic units) gives the apparent charge movement per cotransporter.
Mentions: The slippage mode was modeled as the four-state “iso uni uni” system depicted in 1 A (Segel 1975) and corresponding to states 1, 2, 9, and 10 of the full model in 9. Transitions between states were modeled according to the Eyring–Boltzmann transition rate theory (Adrian 1978). We assumed that the empty carrier has a valency, zc = −1, that moves an electrical distance δ through the membrane between the external and internal Na+ binding sites. A single Na+ ion moves an equivalent electrical distance α′ on the cis side and α″ on the trans side to the respective binding sites (Läuger 1991; Parent et al. 1992) so that α′ + δ + α″ = 1. The translocation (step 2 ⇔ 3) with Na+ bound is electroneutral. The eight pseudo first order rate constants, assuming symmetrical energy barriers for each transition, are then given by: \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\begin{matrix}k_{12}={\mathrm{Na}}_{{\mathrm{0}}}k^{0}_{12}{\mathrm{exp}} \left \left(-a{\mathrm{^{\prime}{\mu}}}\right) \right \\ k_{21}=k^{0}_{21}{\mathrm{exp}} \left \left(a{\mathrm{^{\prime}{\mu}}}\right) \right \\ k_{23}=k^{0}_{23}\\ k_{32}=k^{0}_{32}\\ k_{34}=k^{0}_{34}{\mathrm{exp}} \left \left(-a{\mathrm{^{\prime\prime}{\mu}}}\right) \right \\ k_{43}={\mathrm{Na}}_{{\mathrm{i}}}k^{0}_{43}{\mathrm{exp}} \left \left(a{\mathrm{^{\prime\prime}{\mu}}}\right) \right \\ k_{14}=k^{0}_{14}{\mathrm{exp}} \left \left({\mathrm{{\delta}{\mu}}}\right) \right \\ k_{41}=k^{0}_{41}{\mathrm{exp}} \left \left(-{\mathrm{{\delta}{\mu}}}\right) \right \end{matrix}\end{equation*}\end{document} and μ = eV/2kT, where, Nao and Nai are the external and internal Na+ concentrations (M), respectively, V is the transmembrane voltage, e is the electronic charge, k is Boltzmann's constant, T is the absolute temperature, and k012, k021, etc., are the rate constants at V = 0.

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