In the search of the flip state of ligand gated receptors: building the appropriate solution switching system for its detection.

AUZMENDI, JERÓNIMO A; MANTILLA, MIGUEL; LÓPEZ, LUCÍA; PALLAVECINI, CARLA; PIEAGARI, ESTEFANÍAE; MOFFATT, LUCIANO

Bahia Blanca, Pcia Buenos Aires

Workshop; Neuronal Communication: From structure to physiology; 2008

Sociedad Argentina de Investigación en Neurociencias

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> The hypothesis of a flip state, intermediate between the fully liganded but closed receptor and the open receptor, solves two problems at once. On one hand it explains the fact that the association rate of each binding site of the receptor depends on the occupation state of neighbor sites. It also lifts the requirement of a concerted movement at the gate and the binding sites each time the channel switches between the open and the non desensitized closed state. A flip state has been detected in P2X2 as a 0.1 ms delay in the opening of the channel upon the exposure of 0.2 ms pulse of the agonist. The detection of the flip state in other ligand gated receptors requires of briefer applications. Two technical difficulties have limited the time reduction in the drug application in patch clamp experiments on the outside out configuration. The first is the ability of moving the interface between the drug-free and drug rich solutions fast enough. The second is the ability of reducing the thickness of the unstirred layer of solution that is in direct contact with the patch membrane. The drug has to cross this layer by diffusion in order to reach the receptor. In present work we present encouraging evidence that such applications are possible. We show that, after building the appropriate power source and a proper optimization, a PZT piezo actuator can move in pulses of 25 microns in 0.02 ms. On the other hand, a solution delivery system is presented that would allow for a substantial reduction in the thickness of the unstirred layer by applying the solution much faster at the time of the solution switch. We show that solenoid pinch valves can control this system in less than 3 ms. The possibilities and limitations of replacing the piezo with a solenoid valve are addressed.