Single-channel analysis of nicotinic acetylcholine receptor alpha4beta2 activation and modulation by Lypd6

BARILA, E.P.; CARIGNANO, C.; SPITZMAUL, G.

Mar del Plata

Congreso; LI Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2015

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

Nicotinic acetylcholine receptor (nAChR) α4β2 is the most abundant nAChRs in the brain, which can be modulated by endogenous molecules such as Lypd6. Our aim is to characterize its activation kinetic and how Lypd6 affects this process. Analysis of channel activation was carried out on cells transiently transfected with α4β2 subunits by patch-clamp in the cell-attached configuration. Acetylcholine (ACh) elicited single-channel activity at all concentrations whose frequency increases with concentration (0.5-100 μM). Two channel amplitudes were recorded, a high (HC) and low (LC) conductance. LC channel predominated at low ACh concentration, meanwhile HC channel became more frequent at higher concentration. Both channel-conductance exhibited 2 open components although duration and activation patterns were different: LC channel gated mostly as single events meanwhile HC channel showed a cluster behavior. Our data suggests that α4β2 assemble with 2 stoichiometries: one with high affinity but low efficacy (LC channel (α4)2(β2)3) and the other with low affinity and high efficacy (HC channel, (α4)3(β2)2). Co-expression of Lypd6 with nAChR yields a 10-fold increase in channel frequency without significant changes in conductance, mean open time or channel activation patterns. Thus indicating Lypd6 modulation of α4β2 by receptor desensitization, channel trafficking or cell membrane stability.