A COMPARATIVE STUDY OF THE PROPERTIES OF a9a10 NICOTINIC RECEPTORS FROM Gallus gallus AND Rattus norvegicus.

LIPOVSEK M; FRANCHINI L; KATZ E; ELGOYHEN AB

Foz do Iguaçu – Brazil

Simposio; XIII International Symposium on Cholinergic Mechanisms; 2008

INTRODUCTION. The a9 and a10 nicotinic acetylcholine receptor (nAChR) subunits are expressed in cochlear hair cells, where they form the receptor that participates in the efferent control of auditory function. It mediates the inhibitory synapse between efferent fibers and outer hair cells in mammals, or short hair cells in birds. The inhibition results from calcium entry through the nAChR, in the presence of acetylcholine (ACh), followed by the activation of a calcium dependent potassium current. Sequence analysis of the CHRNA10 genes suggests that the subunits they encode may present different functional properties when comparing mammalian and non-mammalian species (Franchini, Mol Phylogenet Evol, 41:622, 2006). Our working hypothesis states that the a9a10 receptor from R. norvegicus presents functional properties different from those of the G. gallus receptor. In addition, we propose that those differential properties were acquired during the evolution of the mammalian linage, through the positive selection of non-synonymous substitutions. METHODS. The cDNA encoding the a10 subunit was isolated, from a chicken cochlear cDNA library, through PCR using specific primers. The PCR product was then subcloned into the pS-GEM vector, suitable for expression in Xenopus laevis oocytes. To study the properties of the recombinant chicken a9a10 receptor, we used the two electrode voltage clamp technique on Xenopus oocytes expressing these subunits. We studied the sensitivity to ACh, the desensitization properties and the calcium permeability of the recombinant receptor. RESULTS. The sensitivity to ACh of the G. gallus receptor was similar to that of the R. norvegicus receptor (EC50=21.7±1.2mM and 13.8±1.7mM, respectively). The G. gallusa9a10 receptor presented desensitization properties that resembled more closely those of the a9 than those of the a9a10 R. norvegicus receptor. The calcium permeability of the G. gallus receptor was strikingly lower than that of the mammalian receptor. DISCUSSION. The results presented here support the hypothesis that the mammalian a9a10 receptor acquired new functional properties through the positive selection of non-synonymous substitutions in the a10 subunit during the evolution of this linage.