Fast, Triangular Voltage Clamp for Recording and Kinetic Analysis of an Ion Transporter Expressed in Xenopus Oocytes

Abstract

We present a procedure for determination of 11 system parameters of an ion transporter expressed in Xenopus oocytes. The experiments consist of fast triangular voltage-clamp experiments in the presence and absence of external substrate. A four-state enzymatic cycle operating between an external and an internal section of electrodiffusion is used for analysis. The explicit example treats experiments with the fungal 2 H + - N O 3 − symporter EnNRT, a member of the major superfamily transporters. The results comprise a density of ≈150 fmol functional transporter molecules per oocyte, a gross charge number z E ≈ −0.3 of the empty binding site of the enzyme, individual rate constants for reorientation of the empty and occupied binding site in the range of 5–500 s −1, electrical access sections between bulk solutions and reaction cycle of ∼3% inside and 15% outside, an increase of internal N O 3 − at the plasma membrane from ∼0.5 to ∼2 mM during exposure to external N O 3 − , and K D ≈ 0.3 μM 3 inside and K D ≈ 3 μM 3 outside in binding the triplicate substrate ( 2 H + + N O 3 − ). The results compare well with the known structure of the lactose permease, another major superfamily transporter.