Fast, triangular voltage clamp for recording and kinetic analysis of an ion transporter expressed in Xenopus oocytes

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 2H+-NO3- symporter EnNRT, a member of the major superfamily transporters. The results comprise a density of approximately 150 fmol functional transporter molecules per oocyte, a gross charge number z(E) approximately -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 approximately 3% inside and 15% outside, an increase of internal NO3- at the plasma membrane from approximately 0.5 to approximately 2 mM during exposure to external NO3-, and K(D) approximately 0.3 microM3 inside and K(D) approximately 3 microM3 outside in binding the triplicate substrate (2H+ +NO3-). The results compare well with the known structure of the lactose permease, another major superfamily transporter.