INGEBI   02650
INSTITUTO DE INVESTIGACIONES EN INGENIERIA GENETICA Y BIOLOGIA MOLECULAR "DR. HECTOR N TORRES"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The development of an agonist for the ¨¢9¨¢10 nicotinic cholinergic receptor
Autor/es:
REIFF R; BOFFI JC; PEREZ E; ELGOYHEN AB
Lugar:
Huerta Grande Cordoba
Reunión:
Congreso; II Reunión Conjunta de la SAN y TAN, IIRCN; 2010
Resumen:
The ¦Á9¦Á10 nicotinic acetylcholine receptor (nAChR) is expressed in the outer hair cells (OHCs) of the organ of Corti, where it receives efferent innervation from the medial olivocochlear (MOC) system and plays an important role in the central nervous system¡¯s modulation of sound detection and amplification.  When ACh released by MOCs binds to ¦Á9¦Á10, an influx of Ca2+ is induced.  The subsequent increase in cytosolic calcium leads to activation of Ca2+-dependent SK2 K+ channels, causing a significant efflux of potassium.  The net hyperpolarization of OHCs causes an inhibition of sound amplification, leading to an overall reduction in cochlear sensitivity.  The ¦Á9¦Á10 receptor exhibits an unusual pharmacological profile with mixed muscarinic-nicotinic properties, and with the exception of ACh, no effective agonists are known to exist.  The development of a potent and selective agonist for ¦Á9¦Á10 is extremely important, as increased activity of ¦Á9¦Á10 may be useful in the prevention of noise-induced hearing loss and the treatment of certain auditory processing disorders and learning disabilities. Using information from previous investigations on the molecular dynamics of the ¦Á9¦Á10 binding site, a series of structural acetylcholine analogs were synthesized. In this study, the novel compounds were tested as potential agonists for ¦Á9¦Á10 and characterized through a comparison to the effects of ACh.    Methods Full-length rat ¦Á9 and ¦Á10 cRNA constructs were expressed in Xenopus laevis oocytes, which were maintained in Barth¡¯s solution at 17¡ãC.  Electrophysiological recordings were performed 3-7 days following cRNA injection using the two-electrode voltage-clamp technique.  When recorded currents were high due to activation of the oocyte¡¯s native Ca2+-sensitive chloride channels, cells were incubated for 2-3 hours before recordings with the calcium chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N¡¯,N¡¯-tetraacetic acid-acetoxymethyl ester (BAPTA-AM, 100 ¦ÌM). Oocytes were clamped at approximately -70 mV and perfused with Ringer¡¯s solution.  Drug applications were performed at 1 ¦ÌM, 10 ¦ÌM, 30 ¦ÌM, 100 ¦ÌM, 300 ¦ÌM, 1 mM, and 3 mM concentrations. Dose-response curves were constructed by normalizing all responses to the maximal response of the compound analyzed in order to approximate an EC50.  Compounds were also normalized to the maximal response of ACh, in order to estimate the efficacy of the compound.   Conclusion and Future Directions In this study, four quaternary ammonium salts elicited agonist responses in ¦Á9¦Á10, while the tertiary ammonium compound had no effect.  At high concentrations, two compounds elicited responses greater than the maximal response of ¦Á9¦Á10 to ACh.  One particularly effective compound, 2-((3-methoxy-3-oxopropanoyl)oxy)-N,N,N-trimethylethanaminium iodide (EP124), produced nearly 150% of the maximal response to ACh.  2-((4-methoxy-4-oxobutanoyl)oxy)-N,N,N-trimethylethanaminium iodide (EP126) also produced responses greater than the maximal response to ACh.  Meanwhile, the largest quaternary compound, 2-((5-methoxy-5-oxopentanoyl)oxy)-N,N,N-trimethylethanaminium iodide (EP128) and the smallest quaternary compound, 2-(2-methoxy-2-oxoacetoxy)-N,N,N-trimethylethanaminium iodide (EP123-B), acted as much weaker agonists.  The compound 2-((3-methoxy-3-oxopropanoyl)oxy)-N,N,N-trimethylethanaminium iodide (EP124) has been selected as a lead compound that will now be structurally modified in order to improve selectivity and potency of the agonist.  Future variations of the agonist will be tested against ¦Á9¦Á10 in Xenopus oocytes, as well as native hair cells.  Furthermore, the agonist will need to be tested for effects on other nicotinic cholinergic receptors.  These results indicate a promising step toward the development of a crucial pharmaceutical agent to target the ¦Á9¦Á10 receptor.      References             Elgoyhen AB, Johnson DS, Boulter J, Vetter DE, & Heinemann S. ¦Á9: An acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell 1994;79:705-715.   Elgoyhen AB, Katz E, & Fuchs P. The nicotinic receptor of cochlear hair cells: a possible pharmacotherapeutic target? Biochem Pharmacol 2009;78:712-9.   Elgoyhen AB, Vetter D, Katz E, Rothin C, Heinemann S, & Boulter J. ¦Á10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc Natl Acad Sci USA 2001;98:3501-6.   P¨¦rez EG, Cassels BK, & Zapata-Torres G. Molecular modeling of the ¦Á9¦Á10 nicotinic acetylcholine receptor subtype. Bioorch Med Chem Lett 2009;19:251-254.