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

¹ Abtlg. Biophysik, AvH-Inst. f. Pflanzenwiss.
² Department of Oceanography, Dalhousie University Halifax, NS

^* To whom correspondence should be addressed. E-mail: dgradma{at}gwdg.de.

Submitted on February 4, 2005
Revised on February 28, 2005
Accepted on 14 April 2005

	Abstract

We present a procedure for determination of eleven 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 (MSF) transporters. The results comprise a density of 150 fmol functionaltransporter 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 about 3 % inside and 15 % outside, an increase of internal NO₃^- at the plasma membrane from about 0.5 to about 2 mM during exposure to external NO₃^-, and K_D's 0.3 µM³ inside and 3 µM³ outside in binding the triplicate substrate (2H⁺ + NO₃^-). The results compare well with the known structure of the lactose permease, another MSF transporter.

Key Words: MSF transporters, difference current-voltage curves, enzyme kinetics, symport, transporter turnover, triangular voltage-clamp