Pannexin1 expression in Xenopus laevis oocytes

GALIZIA L, ALLEVA K. SCHWARZBAUM PJ

Simposio; Reunión Conjunta de Sociedades de Biociencias; 2017

Six subunits of Pannexin 1 (Panx1) form a plasma membranechannel termed ?pannexon?. The pannexon can function as asmall-conductance chloride selective channel, activated by an increaseof membrane potential (Vm). Alternatively, a non selectivehigh- conductance mode was reported, where the channel can beactivated by increases in extracellular potassium concentration (K+)e and permeate molecules less than 1.5 kD. In addition to K+e andVm, hypotonicity was suggested ?but not thoroughly proven- to activatePanx1 activity.In this context we used Xenopus oocytes as an expression systemto express Panx1, and characterize the modulation of Panx1 currentsby K+e and hypotonicity.Expression of Panx1 was assessed by two-electrode voltageevaluatethe small conductance mode or a continuous pulse protocol toobserve the high conductance mode. By applying a voltage stepprotocol in Panx1 injected oocytes we observed voltage sensitiveoutward currents that were absent in water injected oocytes (Ipanx1 at60 mV = 0.60 ± 0.12 μA, n=10 vs Iwater at 60 mV 0.12 ± 0.01μA, n=7,p<0.05), while addition of CBX (a Panx1 inhibitor) suppressed thesecurrents. Assessment of Panx1 by (K+)e stimulation was performedin a continuous pulse protocol. A 5-fold increase of currents wasevidenced in Panx1 injected oocytes, suggesting the presence ofhigh conductance mode. On the other hand, hypotonic medium (50mOSM, 1.5 min), as compared to isotonic control, did not alter themagnitude of currents (Iiso 60 mV = 0.31 ± 0.12 μA vs Ihypo 0.33 ± 0.12μA, n=5, NS). Results suggest that (K+)e, but not hypotonicity, wasable to activate Panx1 into the high conductance mode of pannexon.This data could be relevant in the understanding of the Pannexin 1role during cell volume changes.With grant from ANPCyT PICT 2014 Nº 0357Keywords: Pannexin 1, Voltage Clamp, Xenopus oocytes, Potassium,Osmotic stimuli