Ultrafast Applications For Determining the Kinetics Of Ligand-gated Channels.

AUZMENDI, JERÓNIMO A; LÓPEZ, LUCÍA; PALLAVECINI, CARLA; PIEAGARI, ESTEFANÍA; RADUSKY, LEANDRO; MOFFATT, LUCIANO

Boston, MA, USA

Congreso; Biophysical Society Annual meeting; 2009

Biophysical Society

Determining kinetic models that describe and predict the behavior of ligand-gated channels is a daunting task. It can be done by the analysis of a considerable amount of single channel recordings or, as it has recently shown for P2X2 receptors, by the analysis of macrocurrents after the application of very short pulses of different concentrations of the agonist. Here we present some advances towards a general strategy for determining a kinetic model for any particular ligand-gated channel using ultrashort pulses. First is the problem of generating the agonist pulses. For that purpose we optimize the movement of the liquid interface between agonist solution and saline. This movement is generated by driving a PZT actuator, whose movement is measured by the deflection of a laser that hits a four quadrant movement detector. The movement of the piezo was optimized to produce pulses as large as 20 micrometers and as brief as 20 microseconds of duration. The piezo drives the movement of a theta tube that eject the agonist and the saline, the movement of whom is measured using stroboscopic microscopy. On the other hand, in order to achieve high speed of solution exchange, a high velocity of fluid is necessary at the right times, so a valve-controlled pressurized system was developed. On patch controls have to be made by switching from saline to a low cation solution using the drop in current as an indicator of the time profile of agonist concentration as seen by the patch. Finally, the gathered data is analyzed by contrasting the likelihood of different allosteric models. For that purpose, an extension of the Macroscopic Recursive method is presented that allow its application to time averaged recordings.