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Structure-function relations of the first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter: II. Substrate interaction and voltage dependency of two functionally important sites.

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

Bottom Line: At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification.The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics.The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

ABSTRACT
Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

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Voltage dependency of Na+ activation in 1 mM Pi. (A) Na+ activation characteristics for WT-expressing oocytes determined at six membrane potentials from −100 to 0 mV as indicated by different symbols. Each data point is the difference between current recorded in NDX + Pi and NDX, shown as mean ± SEM (n = 4), where X is the respective Na+ concentration (in mM) and Pi = 1 mM. Currents were normalized to the magnitude of IPi at 1 mM Pi, V = −100 mV, and ND100. Arrow indicates direction of depolarization. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter. (B) Normalized Na+ activation data for G134C-expressing oocytes determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for G134C + MTS, and nH = 2 for G134C − MTS. Note the different ordinate scales for the −MTS and +MTS cases. Currents were normalized to the magnitude of IPi for G134C + MTS, ND100, V = −100 mV. (C) Normalized Na+ activation data for M533C-expressing oocytes (n = 4) determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for M533C − MTS, and nH = 2 for M533C + MTS. Note the different ordinate scales for the −MTS and +MTS cases. Currents were normalized to the magnitude of IPi for M533C − MTS, ND100, V = −100 mV. (D) Voltage dependency of apparent Na+ affinity (KmNa) for G134C (left) and M533C (right). Data points indicate mean ± SEM of KmPi reported from fits to Na+ activation data for the individual oocytes pooled in A–C. WT, filled circles; mutant − MTS, filled squares; mutant + MTS, empty squares.
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fig2: Voltage dependency of Na+ activation in 1 mM Pi. (A) Na+ activation characteristics for WT-expressing oocytes determined at six membrane potentials from −100 to 0 mV as indicated by different symbols. Each data point is the difference between current recorded in NDX + Pi and NDX, shown as mean ± SEM (n = 4), where X is the respective Na+ concentration (in mM) and Pi = 1 mM. Currents were normalized to the magnitude of IPi at 1 mM Pi, V = −100 mV, and ND100. Arrow indicates direction of depolarization. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter. (B) Normalized Na+ activation data for G134C-expressing oocytes determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for G134C + MTS, and nH = 2 for G134C − MTS. Note the different ordinate scales for the −MTS and +MTS cases. Currents were normalized to the magnitude of IPi for G134C + MTS, ND100, V = −100 mV. (C) Normalized Na+ activation data for M533C-expressing oocytes (n = 4) determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for M533C − MTS, and nH = 2 for M533C + MTS. Note the different ordinate scales for the −MTS and +MTS cases. Currents were normalized to the magnitude of IPi for M533C − MTS, ND100, V = −100 mV. (D) Voltage dependency of apparent Na+ affinity (KmNa) for G134C (left) and M533C (right). Data points indicate mean ± SEM of KmPi reported from fits to Na+ activation data for the individual oocytes pooled in A–C. WT, filled circles; mutant − MTS, filled squares; mutant + MTS, empty squares.

Mentions: Fig. 2 shows the Na+ activation data at 1 mM Pi for oocytes that expressed WT (A), G134C (B), and M533C (C). Each panel depicts the Pi-dependent current (IPi) at different membrane potentials in the range −100 ≤ V ≤ 0 mV. As for the Pi activation (Fig. 1), the normalized and pooled data for G134C and M533C are shown before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. The data for G134C + MTS, M533C − MTS, and the WT displayed a similar pattern with a strong voltage dependency at the maximum Na+ (125 mM) used in these assays. For G134C − MTS and M533C + MTS, the relative Pi currents were suppressed compared with G134C + MTS and M533C − MTS, respectively, and in all cases IPi showed evidence of saturation with increasing Na+ concentration. Like the Pi activation data, there was an inverse relationship with voltage for the maximum dependent current, and, moreover, a reversal of IPi was observed for G134C − MTS and M533C + MTS at low Na+ concentrations. The data were fit with a form of the modified Hill equation with a variable offset (Eq. 2) to estimate the apparent Na+ affinity for cotransport (KmNa) (Fig. 2 D). Reliable fits of Eq. 2 to the WT, G134C + MTS, and M533C − MTS data were obtained with nH as a free parameter. We found that nH was reasonably constant when averaged over the estimates at each test potential (WT, 2.0 ± 0.1; G134C + MTS, 1.8 ± 0.1; M533C − MTS, 2.2 ± 0.2). These data confirmed a cooperative Na+ interaction with the engineered transporters and suggested that for G134C + MTS, the cooperativity was possibly reduced compared with the WT. For the G134C − MTS and M53C + MTS data, where the currents were typically <−30 nA, reliable fits of Eq. 2 over the same voltage range were only obtained by constraining nH = 2.


Structure-function relations of the first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter: II. Substrate interaction and voltage dependency of two functionally important sites.

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

Voltage dependency of Na+ activation in 1 mM Pi. (A) Na+ activation characteristics for WT-expressing oocytes determined at six membrane potentials from −100 to 0 mV as indicated by different symbols. Each data point is the difference between current recorded in NDX + Pi and NDX, shown as mean ± SEM (n = 4), where X is the respective Na+ concentration (in mM) and Pi = 1 mM. Currents were normalized to the magnitude of IPi at 1 mM Pi, V = −100 mV, and ND100. Arrow indicates direction of depolarization. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter. (B) Normalized Na+ activation data for G134C-expressing oocytes determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for G134C + MTS, and nH = 2 for G134C − MTS. Note the different ordinate scales for the −MTS and +MTS cases. Currents were normalized to the magnitude of IPi for G134C + MTS, ND100, V = −100 mV. (C) Normalized Na+ activation data for M533C-expressing oocytes (n = 4) determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for M533C − MTS, and nH = 2 for M533C + MTS. Note the different ordinate scales for the −MTS and +MTS cases. Currents were normalized to the magnitude of IPi for M533C − MTS, ND100, V = −100 mV. (D) Voltage dependency of apparent Na+ affinity (KmNa) for G134C (left) and M533C (right). Data points indicate mean ± SEM of KmPi reported from fits to Na+ activation data for the individual oocytes pooled in A–C. WT, filled circles; mutant − MTS, filled squares; mutant + MTS, empty squares.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234003&req=5

fig2: Voltage dependency of Na+ activation in 1 mM Pi. (A) Na+ activation characteristics for WT-expressing oocytes determined at six membrane potentials from −100 to 0 mV as indicated by different symbols. Each data point is the difference between current recorded in NDX + Pi and NDX, shown as mean ± SEM (n = 4), where X is the respective Na+ concentration (in mM) and Pi = 1 mM. Currents were normalized to the magnitude of IPi at 1 mM Pi, V = −100 mV, and ND100. Arrow indicates direction of depolarization. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter. (B) Normalized Na+ activation data for G134C-expressing oocytes determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for G134C + MTS, and nH = 2 for G134C − MTS. Note the different ordinate scales for the −MTS and +MTS cases. Currents were normalized to the magnitude of IPi for G134C + MTS, ND100, V = −100 mV. (C) Normalized Na+ activation data for M533C-expressing oocytes (n = 4) determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for M533C − MTS, and nH = 2 for M533C + MTS. Note the different ordinate scales for the −MTS and +MTS cases. Currents were normalized to the magnitude of IPi for M533C − MTS, ND100, V = −100 mV. (D) Voltage dependency of apparent Na+ affinity (KmNa) for G134C (left) and M533C (right). Data points indicate mean ± SEM of KmPi reported from fits to Na+ activation data for the individual oocytes pooled in A–C. WT, filled circles; mutant − MTS, filled squares; mutant + MTS, empty squares.
Mentions: Fig. 2 shows the Na+ activation data at 1 mM Pi for oocytes that expressed WT (A), G134C (B), and M533C (C). Each panel depicts the Pi-dependent current (IPi) at different membrane potentials in the range −100 ≤ V ≤ 0 mV. As for the Pi activation (Fig. 1), the normalized and pooled data for G134C and M533C are shown before (top) and after (bottom) incubation in 1 mM MTSEA for 3–5 min. The data for G134C + MTS, M533C − MTS, and the WT displayed a similar pattern with a strong voltage dependency at the maximum Na+ (125 mM) used in these assays. For G134C − MTS and M533C + MTS, the relative Pi currents were suppressed compared with G134C + MTS and M533C − MTS, respectively, and in all cases IPi showed evidence of saturation with increasing Na+ concentration. Like the Pi activation data, there was an inverse relationship with voltage for the maximum dependent current, and, moreover, a reversal of IPi was observed for G134C − MTS and M533C + MTS at low Na+ concentrations. The data were fit with a form of the modified Hill equation with a variable offset (Eq. 2) to estimate the apparent Na+ affinity for cotransport (KmNa) (Fig. 2 D). Reliable fits of Eq. 2 to the WT, G134C + MTS, and M533C − MTS data were obtained with nH as a free parameter. We found that nH was reasonably constant when averaged over the estimates at each test potential (WT, 2.0 ± 0.1; G134C + MTS, 1.8 ± 0.1; M533C − MTS, 2.2 ± 0.2). These data confirmed a cooperative Na+ interaction with the engineered transporters and suggested that for G134C + MTS, the cooperativity was possibly reduced compared with the WT. For the G134C − MTS and M53C + MTS data, where the currents were typically <−30 nA, reliable fits of Eq. 2 over the same voltage range were only obtained by constraining nH = 2.

Bottom Line: At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification.The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics.The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

ABSTRACT
Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

Show MeSH
Related in: MedlinePlus