Ion Accumulation and Depletion in Patch Clamp Experiments

Abstract

The flow of ions through channels and transporters depends sensitively on the concentration of the ionic species and voltage at the membrane. In the widely used patch-clamp technique, voltage and current are measured using electrodes in the pipette and bath that are distant from the membrane. Although the importance of voltage differences between the membrane and electrodes is widely appreciated, concentration gradients arising from the flow of ions are often neglected. In this study, we modeled changes in voltage and ion concentrations during patch-clamp experiments using the Nernst-Plank equation and we derived simple formulas for estimating the timescale and extent of ion accumulation and depletion. For excised patch experiments, ions crossing the membrane can directly diffuse into or out of the patch pipette and ion concentrations stabilize on the millisecond timescale after a change in membrane current. In contrast, in whole-cell experiments the cytosol acts as a reservoir and ion concentrations change on the timescale of seconds. In either configuration, ion accumulation or depletion at steady-state depends primarily on the electrode access resistances and currents carried by each ionic species. As a practical illustration, simulations were performed for bi-ionic protocols previously used to characterize the dynamic ionic selectivity of P2X and TRPV channels. Importantly, even when the net current was small and the membrane voltage effectively clamped, ion accumulation and depletion could cause significant, time-dependent changes in current resembling reported examples of “pore dilation”. Thus, limitations for clamping ion concentrations should be considered when performing and interpreting patch-clamp experiments.