The effect of proteolytic enzymes on the alpha 9-nicotinic receptor-mediated response in isolated frog vestibular hair cells

In frog vestibular organs, efferent neurons exclusively innervate type II h air cells. Acetylcholine, the predominant efferent transmitter, acting on a cetylcholine receptors of these hair cells ultimately inhibits and/or facil itates vestibular afferent firing. A coupling between alpha9-nicotinic acet ylcholine receptors (alpha 9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) is thought to drive the inhibiti on by hyperpolarizing hair cells thereby decreasing their release of transm itter onto afferents. The presence of a9nAChR in these cells was demonstrat ed using pharmacological,immunocytochemical, and molecular biological techn iques. However, fewer than 10% of saccular hair cells dissociated using pro tease VIII, protease XXIV, or papain responded to acetylcholine during perf orated-patch clamp recordings. When present, these responses were invariabl y transient, small in amplitude, and difficult to characterize. In contrast , the majority of saccular hair cells (similar to 90%) dissociated using tr ypsin consistently responded to acetylcholine with an increase in outward c urrent and concomitant hyperpolarization, In agreement with alpha 9nAChR ph armacology obtained in other hair cells, the acetylcholine response in sacc ular hair cells was reversibly antagonized by strychnine, curare, tetraethy lammonium, and apamin. Brief perfusions with either protease or papain perm anently abolished the alpha9-nicotinic response in isolated saccular hair c ells. These enzymes when inactivated became completely ineffective at aboli shing the a9-nicotinic response, suggesting an enzymatic interaction with t he alpha 9nAChR and/or downstream effector. The mechanism by which these en zymes render saccular hair cells unresponsive to acetylcholine remains unkn own, but it most likely involves proteolysis of alpha 9nAChR, SK, or both. (C) 2001 Elsevier Science B.V. All rights reserved.