Fecha de publicación: 2012.
Background: Resolving 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 Findings: Solution 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/Significance: Pulses of this duration open the experimental study of the molecular events that occur between the agonist binding and the opening of the channel. © 2012 Auzmendi et al.
Afiliación de los autores: Auzmendi, J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Afiliación de los autores: Fernández Do Porto, D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Afiliación de los autores: Moffatt, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Palabras claves: agonist; analytical parameters; article; channel gating; electric potential; flow rate; measurement; oscillation; patch clamp; piezoelectricity; pipette; process optimization; solution exchange system; Electricity; Movement; Patch-Clamp Techniques; Rheology; Solutions; Time Factors.
Repositorio: Biblioteca Digital (UBA-FCEN). Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales