Effects of pipette geometry on the time course of solution change in patch clamp experiments.
The time course of change in current through KATP channels in inside-out membrane patches, after step change of permeant ion (K+) concentration, was measured. A simple model of the patch as a membrane disc at the base of a cone with the apex removed, was able to describe the time course of channel activity after step change of [K+]. By measuring pipette geometry and using jumps of [permeant ion], it was then possible to estimate the time course of concentration at the membrane for jumps of any other ion or gating ligand. A simple channel block mechanism was used to simulate experiments with concentration jumps of a blocking ligand. The rate constants for ligand-channel interaction were extracted by least-squares fitting of computed mass action responses to those observed in simulated experiments. The simulations showed that even with diffusion delays of hundreds of milliseconds (as may occur in inside-out patch experiments), ligand association and dissociation rates of up to 1,000 s-1 could be accurately extracted by this approach. The approach should be generally applicable to the analysis of ligand concentration jump experiments on any ion channel whose activity is modulated by intracellular ligand.
Cannell
M B
MB
Department of Pharmacology and Clinical Pharmacology, St. George's Hospital Medical School, London, United Kingdom.
Nichols
C G
CG
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D003198:Computer Simulation; D004058:Diffusion; D066298:In Vitro Techniques; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008839:Microelectrodes; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D051381:Rats
D007473:Ion Channels