α9α10 nAChRs: determinants of calcium permeability as selected by nature.

MARCELA LIPOVSEK; LUCÍA FRANCHINI; NEIL MILLAR; PAUL A. FUCHS; ELEONORA KATZ; ANA BELÉN ELGOYHEN

Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

Congreso; Nicotinic Acetylcholine Receptors 2011; 2011

Wellcome Trust Conference Centre

Free Ca2+ ions participate in many biological pathways and regulate various cellular functions. Based on their ability to permeate Ca2+, nicotinic cholinergic receptors (nAChRs) are subdivided into three categories. The muscle type nAChR has a small but significant Ca2+ permeability (PCa/PNa ~ 0.2-0.3), heteromeric neuronal nAChRs have a higher Ca2+ permeability (PCa/PNa ~ 1.5) and the homomeric α7 nAChR has an even greater Ca2+ permeability (PCa/PNa ~ 10-20). The Ca2+ permeability of the mammalian α9α10 nAChR is among the highest (PCa/PNa ~ 10), in accordance with its known function as the source of Ca2+ entry to cochlear hair cells and modulation of sound amplification (Elgoyhen et al., 2001. PNAS. 98: 3501-6). Surprisingly, we now report that the calcium permeability of chicken recombinant α9α10 nAChRs expressed in Xenopus oocytes (PCa/PNa < 2, n = 8) is much lower than that of its mammalian counterpart and similar to that of α4β2 receptors. This may follow from the differential evolutionary history recently described for mammalian versus non-mammalian vertebrate α9α10 nAChRs (Franchini and Elgoyhen, 2006. Molecular Phylogenetics and Evolution. 41:622-634) and provides the opportunity to analyze the determinants of Ca2+ permeability by searching for the differences in the amino acid sequence that natural selection (and not arbitrary experimental mutagenesis) has fixed. Through the generation of chimeric receptors and site directed mutagenesis, where we have interchanged rat for chicken amino acid residues and vice versa, we demonstrate that transmembrane region 2 of the receptor does not bear the molecular determinants for the differences in the permeability to this cation. Rather, we show that the intracellular ring, located in the TM1-TM2 loop, and residues D/N110 and S/F127, in the extracellular vestibule, are intimately involved in determining the Ca2+ permeability of α9α10 nAChRs. In addition, selectivity determinants are also present in the intracellular loop. These results do not support the notion that transmembrane region 2 is solely responsible for the calcium permeability of nAChRs, but rather support recent findings showing that ions are selected along the entire conduction pathway (Peters et al., 2010. J Physiology. 588.4: 587–595).