Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.
Highlights
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
Estimation of NBCe2-C transport stoichiometry with the conventional reversal potential method The light microscopic image of cultured HEK-293 cells and corresponding fluorescent image of the same field is shown in Figure 1a and b respectively
There is no significant HCO3−-induced current detected in these cells (n = 4). These results indicate that functional NBCe2-C is expressed in Enhanced Green Fluorescent Protein (EGFP) labeled HEK-293 cells and that NBCe2-C transports HCO3− electrogenically
Summary
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. Erev is a complicated function of the sum of different conductances rather than being additive Based on their electrical properties, membrane protein transporters are classified as being either electrogenic (transport a net charge) or electroneutral [1,2,3]. Which of these categories a given transporter belongs to is dependent on its substrate (or ion) coupling ratio; its transport stoichiometry represented by the symbol q. Certain transporters have variable stoichiometry ratios [6,7,8,9,10]
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