Mutational analisys of the agonist binding site of the alpha9 alpha10 nicotinic cholinergic receptor.

BOFFI JC; SAVINO J; LIPOVSEK M; BALLESTERO J; KATZ E; ELGOYHEN AB

Huerta Grande, Córdoba

Congreso; I Reunión Conjunta de la Sociedad Argentina de Neurociencias y el Taller Argentino de Neurociencias (IRCN); 2009

SAN_TAN

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} a:link, span.MsoHyperlink {color:blue; text-decoration:underline; text-underline:single;} a:visited, span.MsoHyperlinkFollowed {color:purple; text-decoration:underline; text-underline:single;} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> MUTATIONAL ANALYSIS OF THE AGONIST BINDING SITE ON THE a9a10 NICOTINIC CHOLINERGIC RECEPTOR.   Juan Carlos Boffi, Jessica Savino, Paola V. Plazas, Eleonora Katz, Ana Belén Elgoyhen.(boffi@dna.uba.ar) INGEBI-CONICET, Buenos Aires, Argentina   The agonist binding site of nicotinic cholinergic receptors is a pocket of aromatic and hydrophobic residues arranged in six loops at the interface of two adjacent subunits. One of the subunits containing loops A, B & C forms the principal component of the binding site and the adjacent subunit containing loops D, E & F forms the complementary component. Loop C has a disulfide bond between two conserved neighboring cysteines. Loop B has a conserved W149 (Torpedo numbering) associated to agonist binding through cation-pi interactions. Finally, W55 in loop D is one of the few conserved residues in the complementary component. The aim of this study was to analyze the importance of these residues in a9a10 receptor and to characterize the contribution of each subunit to the binding site. Site directed mutagenesis of W149F, W55H and C192S/C193S was carried out in both subunits by using QuickChangeII (Stratagene). ACh-gated currents were recorded in two-electrode voltage-clamped  X. laevis oocytes injected with the mutant subunits cRNAs. C192S/C193S mutations showed a similar shift in EC50 for ACh when present in a9 or a10 (a9a10*: EC50 = 145 ± 1 μM, n=17; a9*a10: EC50 = 147 ± 1 μM, n=8; a9*a10*: EC50 = 402 ± 1 μM, n=6). W149F mutations had a greater increase in EC50 when present in a9 (a9a10*: EC50 = 24 ± 1 μM, n=6; a9*a10: EC50 = 110 ± 1 μM, n=5). This was also seen in W55H mutants (a9a10*: EC50 = 96 ± 1 μM, n=3; a9*a10: EC50 = 717 ± 1 μM, n=3). W149F and W55H mutations show that both subunits are able to form principal and complementary components, but a9 is more efficient. C192S/C193S mutations, when compared to W149F, may indicate that the former are influencing agonist binding and/or its coupling to gating.