INVESTIGADORES
PALLAVICINI Carla
congresos y reuniones científicas
Título:
In the search of the flip state of ligand gated receptors: building the appropriate solution switching system for its detection.
Autor/es:
JERONIMO AUZMENDI; MIGUEL MANTILLA; LUCIA LOPEZ; CARLA PALLAVICINI; ESTEFANÍA PIEGARI; LUCIANO MOFFATT
Lugar:
Bahia Blanca
Reunión:
Workshop; Neuronal Communication: From structure to physiology; 2008
Institución organizadora:
Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB)
Resumen:
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.