Recombinant alpha9alpha10 nicotinic cholinergic receptors depend on extracellular Ca2+ to reach maximum activation.

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

Birmingham

Congreso; congress of the International Union of Physiological Sciences, IUPS; 2013

International Union of Physiological Sciences

The activation of alpha9alpha10 nicotinic receptors in cochlear hair cells can ameliorate acoustic trauma. Therefore, maximized alpha9alpha10 receptor activation may favor the prevention of noise-induced hearing loss. Hence, understanding the conditions in which alpha9alpha10 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 alpha9alpha10 receptors expressed in X. laevis oocytes under two-electrode voltage-clamp. Previous work from our lab suggests that the homomeric α9 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 α9α10 receptor, as opposed to homomeric α9, 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 alpha10 subunit provides the structural determinants. In order to delineate these structural determinants, we constructed chimeric alpha10 subunits. Receptors with chimeric alpha10 subunits bearing the alpha9 extracellular and the α10 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 alpha9 transmembrane regions facing the extracellular domain (the TM2-TM3 loop and extracellular segment of TM1) in alpha10 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 alpha9alpha10 receptor activation and that the structural determinant of this effect would be the interphase between extracellular and transmembrane domains of alpha10 subunits. These results also place that region as a possible pharmacological target for noise induced hearing loss.