Achieving solution exchange in the tens of microseconds range for patch clamp experiments

AUZMENDI, JERÓNIMO; FERNÁNDEZ DO PORTO, DARÍO; MOFFATT, LUCIANO

Vancouver

Congreso; 10th Annual Ion Channel Retreat; 2012

The removal of the agonist before a ligand gated channel opens occurs would allow resolving the kinetics of agonist binding events without the interference of subsequent channel gating processes. To study study nicotinic ACh receptor or AMPA receptor activation it would be necessary to apply pulses pulses shorter than 100 microsegundos. No reported system has yet achieved. By moving a sharp interface between a control and an experimental solution across a channel preparation, solution exchange systems deliver short agonist pulses. 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 microsegundos and exchanging 93 ± 1% of the solution, as measured in the open tip of a patch pipette. Pulses of this duration open the experimental study of the molecular events that occur between the agonist binding and the opening of the channel.