Recombinant á9á10 nicotinic cholinergic receptors depend on extracellular Ca2+ to reach maximum activation.

JUAN CARLOS BOFFI; PAOLA PLAZAS; MARCELA LIPOVSEK; ELEONORA KATZ; A. BELÉN ELGOYHEN

Birmingham

Congreso; 37th Congress of the International Union of Physiological Sciences; 2013

International Union of Physiological Sciences

The activation of á9á10 nicotinic receptors in cochlear hair cells can ameliorate acoustic trauma. Therefore, maximized á9á10 receptor activation may favor the prevention of noise-induced hearing loss. Hence, understanding the conditions in which á9á10 receptors open 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 á9á10 receptors expressed in X. laevis oocytes under two-electrode voltage-clamp. Previous work from our lab suggests that the homomeric á9 receptor reaches maximum activation at trace concentrations of extracellular Ca2+ and is blocked by higher concentrations (IC50 = 100±10 ìM, n = 3). We now show that the á9á10 receptor, as opposed to homomeric á9, reaches its lowest maximal currents (at 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 ìM, n = 7; 1.8 mM Ca2+: EC50 = 13±2 ìM, n = 8). This differential dependency upon extracellular Ca2+ suggests that the á10 subunit provides the structural determinants. In order to delineate these structural determinants, we constructed chimeric á10-á9 subunits. Receptors with chimeric á10 subunits bearing the á9 extracellular and the á10 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 á9 transmembrane regions facing the extracellular domain (the TM2-TM3 loop and extracellular segment of TM1) in the chimeras further boosted the activation at trace extracellular Ca2+ (390±40% of maximal currents at 1.8 mM Ca2+; n = 4). Altogether, our results suggest that extracellular Ca2+ greatly affects á9á10 receptor activation and that the structural determinants of this effect are located at the interphase between extracellular and transmembrane domains of the á10 subunits, a region known to be responsible for channel gating.