Functional properties of human alpha7 AChR co-expressed with RIC-3 in a stable neuro-epithelial cell line

VALLÉS, A.S.; ROCCAMO, A.M.; BARRANTES, F.J.

Foz de Iguazú;, Paraná;, Brazil.

Congreso; XIII International Symposium on Cholinergic Mechanisms.; 2008

ISCM

Introduction: a7-Type neuronal nicotinic acetylcholine receptor (AChR) is involved in many physiologically relevant processes in the central nervous system. Relatively little is known about its properties since cell-surface expression of a7AChR in mammalian cells has been very difficult to achieve in the past.  Recently, the chaperone Ric-3 gene has been shown to be necessary for maturation and functional expression of á7AChR. Methods: Transfection of SHEP1-ha7 with hRic-3, clone selection, single–channel patch-clamp recordings and fluorescent and I125aBTX binding assays. Results: Here we describe the production of a stable neuro-epithelial cell line expressing cell-surface human a7AChR when co-transfected with Ric-3 (SHEP1-ha7-Ric3). Electrophysiological recordings in the presence of 50 mM acetylcholine showed characteristic single-channel traces with a main brief opening component. Labeling with [125I]- aBTX or Alexa488-aBTX in the absence or presence of different concentrations of nicotine (0-500 mM) revealed the presence of a7AChR protein in the plasma membrane with a calculated Kd of about 50 nM. [125I]- aBTX or Alexa488-aBTX binding saturated after ~10 min incubation at 4°C. In contrast,  SHEP1-ha7 cells that expressed basal a7AChR levels failed to produce detectable single-channel currents, and did not exhibit measurable levels of Alexa488-aBTX nor [125I]- aBTX binding. Discussion: Our results indicate that SHEP1-ha7-Ric3 is a good model system to study function and regulation of neuronal a7AChR.