A novel delta current method for transport stoichiometry estimation.
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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.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.
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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. |
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Mentions: Cells expressing NBCe1-A were voltage-clamped at -50 mV, and whole-cell currents were recorded when a series of voltage pulses was applied (Figure 5a). Using the same conditions as above that [Na+]i = [Na+]o = 10 mM and [HCO3−]i = [HCO3−]o = 25 mM (patch solution b and bath solution D in Table 1), increasing the Na+ concentration from 10 to 25 mM in the bath solution (bath solution was switched from solution D to solution E of Table 1) increased voltage-dependent current (Figure 5a middle panel). The net current (ΔI) through NBCe1-A induced by changing [Na+]o (right panel of Figure 5a) was obtained by subtracting the current traces in the solution containing 10 mM [Na+]o from those in 25 mM [Na+]o. The current-voltage (I-V) relation of steady-state currents in bath solution containing 10 mM or 25 mM Na+ is shown in Figure 5b). Figure 5c shows ΔI of NBCe1-A vs. membrane voltages. This was the result of operation of Eq. 4 and the currents mediated by other channels and electrogenic transporters were eliminated. Taking ΔIV1 at V = 0 and ΔIV2 at V = 12 mV, we calculated q using Eq. 7 for every cell. We determined q = 1.87 ± 0.062 (n = 6, Figure 5d). The results indicate that the transport stoichiometry ratio of NBCe1-A is 2 HCO3−: 1 Na+ or 1 CO32−: 1 Na+ in HEK-293 cells. This estimate is consistent with our previous results using the conventional reversal potential method with DIDS [25].Figure 5 |
View Article: PubMed Central - PubMed
Affiliation: Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 USA.
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.