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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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Analysis of presteady-state relaxations: charge–voltage (Q–V) data for a representative oocyte expressing G134C (A) and M533C (B). Each point is given by (QON–QOFF)/2, where QON and QOFF are the charges moved for the ON and OFF voltage steps from and to −60 mV, respectively. Errors smaller than symbol size are not shown. Left, superfusion in ND100; right, superfusion in ND0 for the same oocyte. Filled symbols, before MTSEA exposure; empty symbols, after MTSEA exposure. Dashed lines have been drawn to indicate the apparent equality of charge movement at hyperpolarizing potentials for ND100 and ND0 superfusion for G134C − MTS and M533C + MTS. Continuous lines were obtained by fitting Eq. 3 to the data. For G134C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.1/5.9 nC, Qhyp = −0.9/−3.4 nC; V0.5 = −7/−77 mV; z = 0.5/0.4; and in ND0 (±MTS), Qmax = 4.0/4.3 nC, Qhyp = −1.0/−2.1 nC; V0.5 = −5/−59 mV; z = 0.5/0.4. For M533C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.8/3.7 nC, Qhyp = −2.0/−0.7 nC; V0.5 = −32/+11 mV; z = 0.6/0.6; and in ND0 (±MTS), Qmax = 4.8/2.9 nC, Qhyp = −2.1/−0.6 nC; V0.5 = −48/−5 mV; z = 0.5/0.5.
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fig6: Analysis of presteady-state relaxations: charge–voltage (Q–V) data for a representative oocyte expressing G134C (A) and M533C (B). Each point is given by (QON–QOFF)/2, where QON and QOFF are the charges moved for the ON and OFF voltage steps from and to −60 mV, respectively. Errors smaller than symbol size are not shown. Left, superfusion in ND100; right, superfusion in ND0 for the same oocyte. Filled symbols, before MTSEA exposure; empty symbols, after MTSEA exposure. Dashed lines have been drawn to indicate the apparent equality of charge movement at hyperpolarizing potentials for ND100 and ND0 superfusion for G134C − MTS and M533C + MTS. Continuous lines were obtained by fitting Eq. 3 to the data. For G134C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.1/5.9 nC, Qhyp = −0.9/−3.4 nC; V0.5 = −7/−77 mV; z = 0.5/0.4; and in ND0 (±MTS), Qmax = 4.0/4.3 nC, Qhyp = −1.0/−2.1 nC; V0.5 = −5/−59 mV; z = 0.5/0.4. For M533C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.8/3.7 nC, Qhyp = −2.0/−0.7 nC; V0.5 = −32/+11 mV; z = 0.6/0.6; and in ND0 (±MTS), Qmax = 4.8/2.9 nC, Qhyp = −2.1/−0.6 nC; V0.5 = −48/−5 mV; z = 0.5/0.5.

Mentions: Fig. 6 A shows the charge–voltage (Q–V) relationship for a representative G134C-expressing oocyte for superfusion in ND100 and ND0 before and after MTS exposure. Each point represents the mean of the absolute charge moved after the ON and OFF transitions.1 Before MTS exposure, saturation was clearly observed at hyperpolarizing potentials for both ND100 and ND0, and, moreover, the charge moved for steps to potentials <Vh was the same for both ND100 and ND0. After MTSEA exposure, the charge distribution showed a clear difference between superfusion in ND100 and ND0 for hyperpolarizing potentials as well as less saturation in this voltage range. The Q–V data for a representative M533C-expressing oocyte showed the opposite behavior (Fig. 6 B). Before MTS exposure there was a small difference in charge movement between ND100 and ND0, and evidence of saturation was observed at both hyperpolarizing and depolarizing extremes. After MTS exposure, there was much stronger saturation for hyperpolarizing steps with equality of charge movement for the two superfusion conditions, as for G134C − MTS. To quantify these changes, the Q–V data were fit with a Boltzmann function (Eq. 3), and the fitting results pooled (n = 4) for each mutant are summarized in Table I. The fitted data showed reasonable agreement between the predicted apparent valency (z) derived from the Q–V (Eq. 3) or τ–V (Eq. 4) fits. This parameter also remained reasonably constant before and after MTS treatment and suggested that the treatment had not altered the intrinsic voltage sensitivity of the protein, but only its voltage dependency. For G134C − MTS and M533C + MTS, the ratio of the predicted hyperpolarizing charge derived from the Boltzmann fit in ND100 to that in ND0 (Qhyp100/Qhyp0) was unity as suggested from the Q–V data. For the G134C + MTS, the ratio >1 confirmed that additional mobile charge was available in the presence of external Na+. For M533C, Qhyp100/Qhyp0 remained close to unity before and after MTS exposure.


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)

Analysis of presteady-state relaxations: charge–voltage (Q–V) data for a representative oocyte expressing G134C (A) and M533C (B). Each point is given by (QON–QOFF)/2, where QON and QOFF are the charges moved for the ON and OFF voltage steps from and to −60 mV, respectively. Errors smaller than symbol size are not shown. Left, superfusion in ND100; right, superfusion in ND0 for the same oocyte. Filled symbols, before MTSEA exposure; empty symbols, after MTSEA exposure. Dashed lines have been drawn to indicate the apparent equality of charge movement at hyperpolarizing potentials for ND100 and ND0 superfusion for G134C − MTS and M533C + MTS. Continuous lines were obtained by fitting Eq. 3 to the data. For G134C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.1/5.9 nC, Qhyp = −0.9/−3.4 nC; V0.5 = −7/−77 mV; z = 0.5/0.4; and in ND0 (±MTS), Qmax = 4.0/4.3 nC, Qhyp = −1.0/−2.1 nC; V0.5 = −5/−59 mV; z = 0.5/0.4. For M533C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.8/3.7 nC, Qhyp = −2.0/−0.7 nC; V0.5 = −32/+11 mV; z = 0.6/0.6; and in ND0 (±MTS), Qmax = 4.8/2.9 nC, Qhyp = −2.1/−0.6 nC; V0.5 = −48/−5 mV; z = 0.5/0.5.
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fig6: Analysis of presteady-state relaxations: charge–voltage (Q–V) data for a representative oocyte expressing G134C (A) and M533C (B). Each point is given by (QON–QOFF)/2, where QON and QOFF are the charges moved for the ON and OFF voltage steps from and to −60 mV, respectively. Errors smaller than symbol size are not shown. Left, superfusion in ND100; right, superfusion in ND0 for the same oocyte. Filled symbols, before MTSEA exposure; empty symbols, after MTSEA exposure. Dashed lines have been drawn to indicate the apparent equality of charge movement at hyperpolarizing potentials for ND100 and ND0 superfusion for G134C − MTS and M533C + MTS. Continuous lines were obtained by fitting Eq. 3 to the data. For G134C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.1/5.9 nC, Qhyp = −0.9/−3.4 nC; V0.5 = −7/−77 mV; z = 0.5/0.4; and in ND0 (±MTS), Qmax = 4.0/4.3 nC, Qhyp = −1.0/−2.1 nC; V0.5 = −5/−59 mV; z = 0.5/0.4. For M533C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.8/3.7 nC, Qhyp = −2.0/−0.7 nC; V0.5 = −32/+11 mV; z = 0.6/0.6; and in ND0 (±MTS), Qmax = 4.8/2.9 nC, Qhyp = −2.1/−0.6 nC; V0.5 = −48/−5 mV; z = 0.5/0.5.
Mentions: Fig. 6 A shows the charge–voltage (Q–V) relationship for a representative G134C-expressing oocyte for superfusion in ND100 and ND0 before and after MTS exposure. Each point represents the mean of the absolute charge moved after the ON and OFF transitions.1 Before MTS exposure, saturation was clearly observed at hyperpolarizing potentials for both ND100 and ND0, and, moreover, the charge moved for steps to potentials <Vh was the same for both ND100 and ND0. After MTSEA exposure, the charge distribution showed a clear difference between superfusion in ND100 and ND0 for hyperpolarizing potentials as well as less saturation in this voltage range. The Q–V data for a representative M533C-expressing oocyte showed the opposite behavior (Fig. 6 B). Before MTS exposure there was a small difference in charge movement between ND100 and ND0, and evidence of saturation was observed at both hyperpolarizing and depolarizing extremes. After MTS exposure, there was much stronger saturation for hyperpolarizing steps with equality of charge movement for the two superfusion conditions, as for G134C − MTS. To quantify these changes, the Q–V data were fit with a Boltzmann function (Eq. 3), and the fitting results pooled (n = 4) for each mutant are summarized in Table I. The fitted data showed reasonable agreement between the predicted apparent valency (z) derived from the Q–V (Eq. 3) or τ–V (Eq. 4) fits. This parameter also remained reasonably constant before and after MTS treatment and suggested that the treatment had not altered the intrinsic voltage sensitivity of the protein, but only its voltage dependency. For G134C − MTS and M533C + MTS, the ratio of the predicted hyperpolarizing charge derived from the Boltzmann fit in ND100 to that in ND0 (Qhyp100/Qhyp0) was unity as suggested from the Q–V data. For the G134C + MTS, the ratio >1 confirmed that additional mobile charge was available in the presence of external Na+. For M533C, Qhyp100/Qhyp0 remained close to unity before and after MTS exposure.

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