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.
Gradmann
Dietrich
D
Abteilung Biophysik der Pflanze der Universität, Untere Karspüle 2, 37073 Göttingen, Germany.
Boyd
Carl M
CM
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D001703:Biophysics / Q000295:instrumentation / Q000379:methods*; D002352:Carrier Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods*; D007477:Ions; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D027981:Symporters / Q000737:chemistry; D013997:Time Factors; D014981:Xenopus
D002352:Carrier Proteins; D007477:Ions; D026901:Membrane Transport Proteins; D027981:Symporters; C021499:lactose permease