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A novel delta current method for transport stoichiometry estimation.

Shao XM, Kao L, Kurtz I - BMC Biophys (2014)

Bottom Line: The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane.Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 USA.

ABSTRACT

Background: The ion transport stoichiometry (q) of electrogenic transporters is an important determinant of their function. q can be determined by the reversal potential (Erev) if the transporter under study is the only electrogenic transport mechanism or a specific inhibitor is available. An alternative approach is to calculate delta reversal potential (ΔErev) by altering the concentrations of the transported substrates. This approach is based on the hypothesis that the contributions of other channels and transporters on the membrane to Erev are additive. However, Erev is a complicated function of the sum of different conductances rather than being additive.

Results: We propose a new delta current (ΔI) method based on a simplified model for electrogenic secondary active transport by Heinz (Electrical Potentials in Biological Membrane Transport, 1981). ΔI is the difference between two currents obtained from altering the external concentration of a transported substrate thereby eliminating other currents without the need for a specific inhibitor. q is determined by the ratio of ΔI at two different membrane voltages (V1 and V2) where q = 2RT/(F(V2 -V1))ln(ΔI2/ΔI1) + 1. We tested this ΔI methodology in HEK-293 cells expressing the elctrogenic SLC4 sodium bicarbonate cotransporters NBCe2-C and NBCe1-A, the results were consistent with those obtained with the Erev inhibitor method. Furthermore, using computational simulations, we compared the estimates of q with the ΔErev and ΔI methods. The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.

Conclusions: We developed a ΔI method for estimating transport stoichiometry of electrogenic transporters based on the Heinz model. This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane. When there are other electrogenic transport pathways, ΔI method eliminates their contribution in estimating q. Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio. This new ΔI method can be readily extended to the analysis of other electrogenic transporters in other tissues.

No MeSH data available.


Computational simulation of membrane currents and reversal potentials. Addition of a Cl− conductance (GCl) has a significant impact on ΔErev and therefore biases the estimation of q of NBC. Based on Eq. 2, currents were calculated with the same conditions as our whole-cell patch-clamp experiments for estimation of q of NBCe2-C and NBCe1-A: [HCO3−]i = [HCO3−]o = 25, [Na+]i =10 mM assuming q = 2 (panels a, b and c) or q = 3 (panels d, e and f). a) I-V curves when bath solution switched from [Na+]o =10 mM to 25 mM and the delta current (ΔI, the dark gray line). b) I-V curves when a relatively small GCl was present (light gray line) and the bath solution switched from [Na+]o =10 mM to 25 mM. C) I-V curves when a relatively larger Cl− conductance (2 x GCl) was present (light gray line) and with the same bath solution switch as b). (d), (e) and (f) show the same operations as (a), (b) and (c) respectively except assuming q = 3. The insets in panel (d), (e) and (f) illustrate VI=0 by enlarging the local areas around I = 0. Y-axis’s are membrane currents of arbitrary unit for comparison purposes.
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Fig6: Computational simulation of membrane currents and reversal potentials. Addition of a Cl− conductance (GCl) has a significant impact on ΔErev and therefore biases the estimation of q of NBC. Based on Eq. 2, currents were calculated with the same conditions as our whole-cell patch-clamp experiments for estimation of q of NBCe2-C and NBCe1-A: [HCO3−]i = [HCO3−]o = 25, [Na+]i =10 mM assuming q = 2 (panels a, b and c) or q = 3 (panels d, e and f). a) I-V curves when bath solution switched from [Na+]o =10 mM to 25 mM and the delta current (ΔI, the dark gray line). b) I-V curves when a relatively small GCl was present (light gray line) and the bath solution switched from [Na+]o =10 mM to 25 mM. C) I-V curves when a relatively larger Cl− conductance (2 x GCl) was present (light gray line) and with the same bath solution switch as b). (d), (e) and (f) show the same operations as (a), (b) and (c) respectively except assuming q = 3. The insets in panel (d), (e) and (f) illustrate VI=0 by enlarging the local areas around I = 0. Y-axis’s are membrane currents of arbitrary unit for comparison purposes.

Mentions: We performed a computational simulation of membrane currents and reversal potentials to show how a conductance in addition to electrogenic NBC transport affects the measurement of VI=0 and thus the estimate of q for this electrogenic NBC. Based on Eq. 2, currents were calculated with the same conditions as our whole-cell patch-clamp experiments for estimating q (delta current method above) of NBCe2-C: [HCO3−]i = [HCO3−]o = 25, [Na+]i =10mM. Assuming q = 2, Figure 6a shows I-V curves and VI=0s when the bath solution switched from [Na+]o =10 mM to 25 mM and the delta current (ΔI). The stoichiometry ratios estimated either with the ΔErev or ΔI methods are equivalent when there was no conductance other than the electrogenic NBC transporter (Table 2). However, if a small Cl− conductance (compared to the conductance of the NBC-mediated current) was present, simulation with Eq. 8 showed that both VI=0 values at [Na+]o = 10 mM and [Na+]o = 25 mM shifted toward more negative value, but the shifts for the two conditions were different (Figure 6b). Therefore ΔErev differed from that obtained without the Cl− conductance and leads to a different estimate of q = 2.17. When the Cl− conductance was doubled, the estimate of q became 2.33 (Figure 6c). When we input q = 3 in the simulation, the estimate was 3 in the absence of any other conductance. After introducing either a small Cl− conductance GCl or 2 x GCl (same as above), the estimate of q became 4.96 and 7.2 respectively with the ΔErev method (Figure 6d,e and f; note the insets; Table 2). However as shown in Table 2, the value of q determined using the ΔI method was unaffected by addition of a GCl on the membrane. Specifically, the ΔI-V curves in the absence, presence of small or large GCl were identical. Therefore, the currents mediated by other channels had been eliminated in the procedure and had no effect on the estimation of q.Figure 6


A novel delta current method for transport stoichiometry estimation.

Shao XM, Kao L, Kurtz I - BMC Biophys (2014)

Computational simulation of membrane currents and reversal potentials. Addition of a Cl− conductance (GCl) has a significant impact on ΔErev and therefore biases the estimation of q of NBC. Based on Eq. 2, currents were calculated with the same conditions as our whole-cell patch-clamp experiments for estimation of q of NBCe2-C and NBCe1-A: [HCO3−]i = [HCO3−]o = 25, [Na+]i =10 mM assuming q = 2 (panels a, b and c) or q = 3 (panels d, e and f). a) I-V curves when bath solution switched from [Na+]o =10 mM to 25 mM and the delta current (ΔI, the dark gray line). b) I-V curves when a relatively small GCl was present (light gray line) and the bath solution switched from [Na+]o =10 mM to 25 mM. C) I-V curves when a relatively larger Cl− conductance (2 x GCl) was present (light gray line) and with the same bath solution switch as b). (d), (e) and (f) show the same operations as (a), (b) and (c) respectively except assuming q = 3. The insets in panel (d), (e) and (f) illustrate VI=0 by enlarging the local areas around I = 0. Y-axis’s are membrane currents of arbitrary unit for comparison purposes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4274721&req=5

Fig6: Computational simulation of membrane currents and reversal potentials. Addition of a Cl− conductance (GCl) has a significant impact on ΔErev and therefore biases the estimation of q of NBC. Based on Eq. 2, currents were calculated with the same conditions as our whole-cell patch-clamp experiments for estimation of q of NBCe2-C and NBCe1-A: [HCO3−]i = [HCO3−]o = 25, [Na+]i =10 mM assuming q = 2 (panels a, b and c) or q = 3 (panels d, e and f). a) I-V curves when bath solution switched from [Na+]o =10 mM to 25 mM and the delta current (ΔI, the dark gray line). b) I-V curves when a relatively small GCl was present (light gray line) and the bath solution switched from [Na+]o =10 mM to 25 mM. C) I-V curves when a relatively larger Cl− conductance (2 x GCl) was present (light gray line) and with the same bath solution switch as b). (d), (e) and (f) show the same operations as (a), (b) and (c) respectively except assuming q = 3. The insets in panel (d), (e) and (f) illustrate VI=0 by enlarging the local areas around I = 0. Y-axis’s are membrane currents of arbitrary unit for comparison purposes.
Mentions: We performed a computational simulation of membrane currents and reversal potentials to show how a conductance in addition to electrogenic NBC transport affects the measurement of VI=0 and thus the estimate of q for this electrogenic NBC. Based on Eq. 2, currents were calculated with the same conditions as our whole-cell patch-clamp experiments for estimating q (delta current method above) of NBCe2-C: [HCO3−]i = [HCO3−]o = 25, [Na+]i =10mM. Assuming q = 2, Figure 6a shows I-V curves and VI=0s when the bath solution switched from [Na+]o =10 mM to 25 mM and the delta current (ΔI). The stoichiometry ratios estimated either with the ΔErev or ΔI methods are equivalent when there was no conductance other than the electrogenic NBC transporter (Table 2). However, if a small Cl− conductance (compared to the conductance of the NBC-mediated current) was present, simulation with Eq. 8 showed that both VI=0 values at [Na+]o = 10 mM and [Na+]o = 25 mM shifted toward more negative value, but the shifts for the two conditions were different (Figure 6b). Therefore ΔErev differed from that obtained without the Cl− conductance and leads to a different estimate of q = 2.17. When the Cl− conductance was doubled, the estimate of q became 2.33 (Figure 6c). When we input q = 3 in the simulation, the estimate was 3 in the absence of any other conductance. After introducing either a small Cl− conductance GCl or 2 x GCl (same as above), the estimate of q became 4.96 and 7.2 respectively with the ΔErev method (Figure 6d,e and f; note the insets; Table 2). However as shown in Table 2, the value of q determined using the ΔI method was unaffected by addition of a GCl on the membrane. Specifically, the ΔI-V curves in the absence, presence of small or large GCl were identical. Therefore, the currents mediated by other channels had been eliminated in the procedure and had no effect on the estimation of q.Figure 6

Bottom Line: The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane.Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 USA.

ABSTRACT

Background: The ion transport stoichiometry (q) of electrogenic transporters is an important determinant of their function. q can be determined by the reversal potential (Erev) if the transporter under study is the only electrogenic transport mechanism or a specific inhibitor is available. An alternative approach is to calculate delta reversal potential (ΔErev) by altering the concentrations of the transported substrates. This approach is based on the hypothesis that the contributions of other channels and transporters on the membrane to Erev are additive. However, Erev is a complicated function of the sum of different conductances rather than being additive.

Results: We propose a new delta current (ΔI) method based on a simplified model for electrogenic secondary active transport by Heinz (Electrical Potentials in Biological Membrane Transport, 1981). ΔI is the difference between two currents obtained from altering the external concentration of a transported substrate thereby eliminating other currents without the need for a specific inhibitor. q is determined by the ratio of ΔI at two different membrane voltages (V1 and V2) where q = 2RT/(F(V2 -V1))ln(ΔI2/ΔI1) + 1. We tested this ΔI methodology in HEK-293 cells expressing the elctrogenic SLC4 sodium bicarbonate cotransporters NBCe2-C and NBCe1-A, the results were consistent with those obtained with the Erev inhibitor method. Furthermore, using computational simulations, we compared the estimates of q with the ΔErev and ΔI methods. The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.

Conclusions: We developed a ΔI method for estimating transport stoichiometry of electrogenic transporters based on the Heinz model. This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane. When there are other electrogenic transport pathways, ΔI method eliminates their contribution in estimating q. Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio. This new ΔI method can be readily extended to the analysis of other electrogenic transporters in other tissues.

No MeSH data available.