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

Dietrich Gradmann ^* and Carl M. Boyd 

^* Abteilung Biophysik der Pflanze der Universität, Untere Karspüle 2, 37073 Göttingen, Germany; and Department of Oceanography, Dalhousie University, Halifax, Nova Scotia, B3H 4J1 Canada

Correspondence: Address reprint requests to Dietrich Gradmann, Tel.: +49-551-39-7838; Fax: +49-551-39-7838; E-mail: dgradma{at}gwdg.de.

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 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 at the plasma membrane from 0.5 to 2 mM during exposure to external and K_D 0.3 µM³ inside and K_D 3 µM³ outside in binding the triplicate substrate (). The results compare well with the known structure of the lactose permease, another major superfamily transporter.