Achieving Maximal Speed of Solution Exchange for Patch Clamp Experiments

Abstract

BackgroundResolving the kinetics of agonist binding events separately from the subsequent channel gating processes requires the ability of applying and removing the agonist before channel gating occurs. No reported system has yet achieved pulses shorter than 100 µs, necessary to study nicotinic ACh receptor or AMPA receptor activation.Methodology/Principal FindingsSolution exchange systems deliver short agonist pulses by moving a sharp interface between a control and an experimental solution across a channel preparation. We achieved shorter pulses by means of an exchange system that combines a faster flow velocity, narrower partition between the two streams, and increased velocity and bandwidth of the movement of the interface. The measured response of the entire system was fed back to optimize the voltage signal applied to the piezoelectric actuator overcoming the spurious oscillations arising from the mechanical resonances when a high bandwidth driving function was applied. Optimization was accomplished by analyzing the transfer function of the solution exchange system. When driven by optimized command pulses the enhanced system provided pulses lasting 26 ± 1 µs and exchanging 93 ± 1% of the solution, as measured in the open tip of a patch pipette.Conclusions/SignificancePulses of this duration open the experimental study of the molecular events that occur between the agonist binding and the opening of the channel.

Catch and Patch: A Pipette-Based Approach for Automating Patch Clamp That Enables Cell Selection and Fast Compound Application

“…If extremely fast compound exchange rates are required, this effect can be minimized by recording from small membrane vesicles instead of whole cells. The theoretical and practical limits of the theta tool method on these so-called outside out recordings have been explored by Auzmendi et al 7 who were able to produce agonist pulses with 8 ms rise time and 26-ms duration. In preliminary experiments, we were able to generate such outside out recordings with our method.…”

“…5 Electrophysiological research on these fast-acting ligand-gated ion channels (LGICs) demands repeated delivery and washout of reagents at a millisecond or even submillisecond timescale. 6,7 To achieve state-of-the-art performance in this discipline, it is required to move the recording electrode with the patchclamped cell away from the bottom of the measurement chamber, where so-called unstirred layer effects slow down the solution exchange, and into the solution stream of a specialized microfluidic device, called a theta tube. 6,8 These devices maintain a laminar flow of two parallel solution streams and use a high performance piezo actuator to switch a cell between the two streams within fractions of a millisecond.…”

Dynamics of T-Junction Solution Switching Aimed at Patch Clamp Experiments

“…In our previous work we applied ligand pulses of 0.2 ms, separating binding from gating on purinergic receptors and we revealed the existence of an intermediate state between them [ 10 ]. Encouraging results of our group [ 11 ] suggest that it would be possible to obtain pulses ten times shorter necessary to resolve this state in nicotinic or glutamatergic receptors in the near future [ 12 , 13 ]. However, in order to understand the role of drugs in channel activity, the application of a single compound at a time is not enough.…”

“…Upstream exchangers are operated with the aid of a solenoid or a pinch valve that alternates the flow from one line to the other. Downstream exchangers either deflect the interface by altering the flow of one of the streams, or move the interface by transversally moving the application device with a piezo or stepper motor [ 10 , 11 ]. Ultra-Fast solution exchanges can be obtained by using downstream exchangers [ 11 ].…”

“…Downstream exchangers either deflect the interface by altering the flow of one of the streams, or move the interface by transversally moving the application device with a piezo or stepper motor [ 10 , 11 ]. Ultra-Fast solution exchanges can be obtained by using downstream exchangers [ 11 ]. By setting a large number of parallel laminar flows and shifting the sample across them it is possible to rapidly switch between 32 solutions [ 26 , 27 ].…”