Kinetics of hyperosmotically‐stimulated Na‐K‐2Cl cotransporter in Xenopus laevis oocytes

A detailed study of hypertonically‐stimulated Na‐K‐2Cl cotransport (NKCC1) in Xenopus laevis oocytes was carried out to understand the 1 K + :1 Cl − transport stoichiometry that was previously observed. We demonstrate that the K + influx through NKCC1 is dependent upon external Na + , even though only a fraction of Na + is co‐transported. In Lineweaver‐Burke plots, all data are best fitted by linear relationships, including Cl − , indicating that only one Cl − ion is transported. We derived the velocity equation for influx under rapid equilibrium and steady‐state assumptions, and demonstrate that the behavior of the family of curves is consistent with a model where Na + binds first, followed by Cl − , then K + . When two Cl − ions bind, the order of binding likely is Cl − , followed by Na + , Cl − , and K + . We show that stimulation of K + movement by K + on the trans side is an intrinsic property of a transporter that carries multiple substrates. Also, variable stoichiometries can be explained by transport reactions involving the translocation of a partially loaded transporter. Furthermore, a smaller transport of Na + , compared to K + , can only be explained if K + releases inside and the transporter moves back outside without releasing Na + . Altogether, our study shows that under hypertonicity, transport through NKCC1 deviates from the normal stoichiometry with one Cl − ion moving, and predicts the same order of ion binding inside and outside.