title: A novel delta current method for transport stoichiometry estimation.
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.
authors.affiliation: Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 USA.
authors.forename: Xuesi M
authors.initials: XM
authors.lastname: Shao
authors.affiliation: Division of Nephrology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 USA.
authors.forename: Liyo
authors.initials: L
authors.lastname: Kao
authors.affiliation: Division of Nephrology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 USA ; Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 USA.
authors.forename: Ira
authors.initials: I
authors.lastname: Kurtz
keywords: Computational simulation; Electrogenic transporter; HEK-293 cells; Membrane current-voltage relationship; Patch clamp; Reversal potential; Stoichiometry