A new delta current method for estimating the transport stoichiometry of electrogenic transporters

Abstract

The stoichiometry of electrogenic transporters is an important determinant of their function. If an electrogenic transporter transports ions in a fixed stoichiometric ratio q, q can be determined by the reversal potential Erev with known intra‐ and extra‐cellular concentrations of transporting ions given that a specific blocker is available. An alternative approach widely used is to calculate delta Erev (ΔErev) by altering concentrations of a transported ion. This approach is based on the hypothesis that the effects of other channels and transporters in cell membrane on Erev are additive. However, the Goldman‐Hodgkin‐Katz (GHK) equation suggests otherwise: Erev is a logarithmic function of the sum of different conductances rather than being additive. In the present study, we propose a delta current (ΔI) method that does not require a blocker. ΔI instead of ΔErev is determined by altering the external concentration of a transported ion thereby eliminating other currents. The ratio of ΔI at two different membrane voltages (E1 and E2) is: ΔI2/ΔI1= (E2/E1) ((exp ((q−1) FE1/RT)−1)/(exp ((q−1) FE2/RT)−1)). ΔI2/ΔI1 only depends on q, E1, and E2. We tested this new ΔI methodology in patch‐clamp experiments of HEK‐293 cells expressing NBC1e1‐A or NBC4c. Our data show that the ΔI equation accurately calculates a stoichiometry ratio of HCO3− :Na+of 2 demonstrating the utility of this new methodology.