Background The ion transport stoichiometry (q) of electrogenic transporters is an

Background The ion transport stoichiometry (q) of electrogenic transporters is an important determinant of their 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 (=?is the sum of all other currents mediated by various channels and electrogenic transporters including leak current on the membrane. can be a non-linear function of V while a general assumption is that it is independent of NBC transport current. If we change the Na+ concentration outside the cell from [Na+]o1 to [Na+]o2 the whole cell current would change from IM1 to IM2. We assume that Kc does not vary with [Na+]o within a range far from saturation. We also assume that the XL647 sum of other currents is a function of XL647 V while the function is unchanged when [Na+]o changes (see Discussion). Therefore the delta current is is completely eliminated. For simplicity we take νNa?=?1 and q?=?νHCO3/νNa. Now we consider at two different voltage points V1 and V2 we have two ΔIM ITGA8 values ΔIV1 and ΔIV2. We take the ratio of them mediated by other channels and transporters in the membrane as a function of V does not change when the substrate concentration is altered [2 15 16 Based on these two assumptions the two methods offer benefits such as experimentally straightforward as changing the concentrations of a substrate without the need for specific blockers and share similar limitations. The difference between ΔI and ΔErev method in terms of assumption 2 is that with the ΔI method can be completely eliminated (Eq.?4) if it does not change when the substrate ([Na+]o in this study) is altered. On the contrary with the ΔErev method as long as is not negligible the confounding effects of on VI=0 can not be eliminated and biases the estimation of q as shown in Figure?6 and Table?2 and Table?3 even if it does not change when the substrate concentration varies. In practice ways to circumvent the limitations due to the above assumptions include: 1) using a smaller concentration change of the substrate as long as it induces a significant delta current; 2) changing the concentrations of a particular substrate with less possibility of involving other electrogenic transporters. For example in the case of electrogenic Na+-coupled glucose or amino acid transporters one would choose to change either glucose or amino acids respectively rather than Na+. In this study we changed [Na+]o from 10 to 25 mM because: 1) HCO3? partakes in a volatile buffer system that involves pCO2 to keep the pH constant. pH would be stable when [HCO3?]o is unaltered; 2) switching [Na+]o from 10 to 25 mM would induce a significant delta current [15] and 3) at these relatively low concentrations the possibility of transport saturation would be small therefore variation of Kc in Eq.?2 and Eq.?3 would be minimized. We assigned V1?=?0 in the above application therefore in the conditions of is well defined and it is not close to 0. In addition we assigned a V2 that is not far from 0 (+12 mV in this study) thus possible variation of Kc under extreme voltages can be minimized. More detailed XL647 kinetic descriptions of the transport rate in order to characterize the entire I-V relationship rely on a detailed understanding of the molecular transport steps [28-30]. This is not necessary for the purposes of our formulation because we implicitly analyze the portion of the I-V relationship that is close to the Erev i.e. V1?=?0 when XL647 [Na+]i?=?[Na+]o and [HCO3?]i?=?[HCO3?]o. The accuracy of stoichiometry estimation using whole-cell patch-clamp recordings also depends on the accuracy of whole-cell current measurement and the voltages applied to the cell membrane from the patch-clamp amplifier. The drift of the junction potential between the patch pipette solution and the Ag/AgCl coated wire that connects to the headstage of the amplifier is a major source of unstable current recording especially when the Cl? concentration in the pipette is low [22]. We used a micro-agar salt bridge of 2 M KCl in the patch pipette that minimized the junction potential drift and therefore stabilized the whole-cell.