INGEBI   02650
INSTITUTO DE INVESTIGACIONES EN INGENIERIA GENETICA Y BIOLOGIA MOLECULAR "DR. HECTOR N TORRES"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Recombinant a9a10 nicotinic cholinergic receptors depend on extracellular Ca2+ to reach maximum activation
Autor/es:
JUAN CARLOS BOFFI; PAOLA PLAZAS; MARCELA LIPOVSEK; ELEONORA KATZ; ANA BELÉN ELGOYHEN
Reunión:
Congreso; 37th congress of the International Union of Physiological Sciences IUPS 2013; 2013
Resumen:
Recombinant a9a10 nicotinic cholinergic receptors depend on extracellular Ca2+ to reach maximum activation. The activation of a9a10 nicotinic receptors in cochlear hair cells can ameliorate acoustic trauma. Therefore, maximized a9a10 receptor activation may favor the prevention of noise-induced hearing loss. Hence, understanding the conditions in which a9a10 receptors reach maximum activation could have a potential therapeutic use in noise-induced hearing loss. In this work we aimed to characterize how extracellular Ca2+ affects the activation of recombinant a9a10 receptors expressed in X. laevis oocytes under two-electrode voltage-clamp. Previous work from our lab suggests that the homomeric a9 receptor reaches maximum activation at trace concentrations of extracellular Ca2+ and is blocked by higher concentrations (IC50 = 100+/-10 uM, n = 3). We now show that the a9a10 receptor, as opposed to homomeric a9, reaches its lowest maximal currents (saturating acetylcholine) at trace levels of extracellular Ca2+ (50+/-15% of maximal currents at physiological 1.8 mM Ca2+; n = 4) and its maximal activation at Ca2+ concentrations close to 0.1mM (260+/-55% of maximal currents at 1.8 mM Ca2+; n = 5). Furthermore, increasing extracellular Ca2+ concentrations reduces the receptor´s EC50 dose dependently (trace Ca2+: EC50 = 54+/-4 uM, n = 7; 1.8 mM Ca2+: EC50 = 13+/-2 uM, n = 8). This differential dependency upon extracellular Ca2+ suggests that the a10 subunit provides the structural determinants. In order to delineate these structural determinants, we constructed chimeric a10 subunits. Receptors with chimeric a10 subunits bearing the a9 extracellular and the a10 transmembrane and intracellular domains reached maximal activation at trace levels of extracelular Ca2+ (190+/-20% of maximal currents at 1.8 mM Ca2+; n = 5). The inclusion of a9 transmembrane regions facing the extracellular domain (the TM2-TM3 loop and extracellular segment of TM1) in a10 subunit chimeras further boosted the activation at trace extracellular Ca2+ (390+/-40% of maximal currents at 1.8 mM Ca2+; n = 4). These results suggest that the structural determinants required for Ca2+ activation might be the interphase between the extracellular and transmembrane domains known to be responsible for channel gating. Altogether, our results suggest that extracellular Ca2+ greatly affects a9a10 receptor activation and that the structural determinant of this effect would be the interphase between extracellular and transmembrane domains of a10 subunits. These results also place that region as a possible pharmacological target for noise induced hearing loss.