Evaluation of single-channel gating kinetics produced after amplitude-based separation of unitary currents.
Cell-attached patch-clamp recording has been established as a major technique for investigating ion channel behaviour in a physiological setting, despite the problems which arise in analysing records containing more than one type of unitary current. To circumvent these problems, single-channel amplitude-based assignment of discrete single-channel events to different channel types becomes increasingly necessary. Surprisingly, a systematic evaluation of the validity of this method in determining single-channel parameters has not been performed to date. Using computer-stimulated single-channel traces, and recordings from a biological preparation containing well-characterized ion channels (N1E-115 neuroblastoma cells), we have explored the accuracy by which amplitude-based separation recovers ion-channel parameters. Determination of gating kinetics after separation revealed that even a very small contamination in the selected population yields additional time constants in the probability density functions. Therefore, our results demonstrate that, whereas the use of amplitude-based separation is straightforward for determining slope conductance and reversal potential, it is prone to incorporate errors in establishing gating kinetics. Ways of identifying such errors are described.
D000818:Animals; D002118:Calcium / Q000502:physiology; D003198:Computer Simulation; D004594:Electrophysiology; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism / Q000502:physiology; D014407:Tumor Cells, Cultured
D015221:Potassium Channels; D002118:Calcium