Analysis of neuronal nicotinic acetylcholine receptors alpha4beta2 activation and modulation by lypd6 at the single-channel level

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

Rio de Janeiro

Congreso; IBRO 9th World Congress 2015; 2015

International Brain Reasearch Organization (IBRO)

Heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) α4β2 are the most abundant and broadly distributed nAChR in the brain. Their function are involved in cognitive processes, nociception and nicotine addiction. α4β2 nAChRs exhibit fast desensitization which hinders characterization of kinetic activation at the single-channel level. Neuronal nAChRs can be modulated by endogenous molecules, such as the membrane-linked proteins, lynx1 or lypd6. Both, the mechanism of action and the nAChR subtypes where lypd6 acts have not yet been determined. Our aim is to characterize kinetic activation of α4β2 receptors and its modulation by lypd6. To analyze channel activation, we used the high-level protein expression cell line, BOSC 23, which was transiently transfected with the neuronal receptor subunits. We subsequently recorded single-channel events of the receptors activated by acetylcholine (ACh) by patch-clamp in the cell-attached configuration. ACh elicits single-channel activity at all concentrations (0.5 to 300 µM). As already reported by others, two different channel amplitudes are observed: a high-(HC) and a low-conductance (LC) channel with a g-70 of 42 and 29 pS, respectively. Channel open frequency varies with the ACh concentration from 0.3 Hz at 0.5 µM to 1.5 Hz at 100 µM ACh. Channel conductance distribution also changes with increasing concentration of ACh. At 0.5 µM ACh, LC openings predominate while at a higher concentration HC openings are more frequent. Mean open-time histograms exhibit two components for LC and HC channels (0.3 and 1.7 ms; and 0.6 and 1.9 ms for LC and HC, respectively). However, at high ACh concentration, HC channels show only one component of ~500 µs. LC channels open mostly as isolated events and less frequently as brief consecutive openings. HC channel activation exhibit several patterns of opening events at 100 µM ACh. It can be observe as: a) isolated events, b) a cluster of 3 to 6 opening events separated by ~100 µs or c) cluster of several openings separated by a closed time of 1.2 ms. α4β2 receptors assemble with 2 stoichiometries, (α4)2(β2)3 and (α4)3(β2)2, which exhibit high- and low-affinity for ACh, respectively. Our single channel analysis suggests that LC and HC channels could be correlated with the high- and low-affinity receptors, respectively. The patterns of channel activity observed for the HC channel indicate that nAChR activation can be generated by the binding of different ACh molecules at non-equivalent binding sites. To analyze the effect of lypd6, we cotransfected its cDNA together with the receptor subunits and analyzed its effects on channel activation at 100 µM ACh. We observed a 10-fold increase in channel frequency in the presence of lypd6 without significant changes in channel conductance, mean open time or channel activation patterns. Our results indicate that lypd6 interacts with α4β2 by modulating receptor desensitization, channel trafficking or stability on cell membrane.