The effect of proteolytic enzymes on the α9-nicotinic receptor-mediated response in isolated frog vestibular hair cells

In frog vestibular organs, efferent neurons exclusively innervate type II hair cells. Acetylcholine, the predominant efferent transmitter, acting on acetylcholine receptors of these hair cells ultimately inhibits and/or facilitates vestibular afferent firing. A coupling between alpha9-nicotinic acetylcholine receptors (alpha 9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) is thought to drive the inhibition by hyperpolarizing hair cells thereby decreasing their release of transmitter onto afferents. The presence of a9nAChR in these cells was demonstrated using pharmacological,immunocytochemical, and molecular biological techniques. However, fewer than 10% of saccular hair cells dissociated using protease VIII, protease XXIV, or papain responded to acetylcholine during perforated-patch clamp recordings. When present, these responses were invariably transient, small in amplitude, and difficult to characterize. In contrast, the majority of saccular hair cells (similar to 90%) dissociated using trypsin consistently responded to acetylcholine with an increase in outward current and concomitant hyperpolarization, In agreement with alpha 9nAChR pharmacology obtained in other hair cells, the acetylcholine response in saccular hair cells was reversibly antagonized by strychnine, curare, tetraethylammonium, and apamin. Brief perfusions with either protease or papain permanently abolished the alpha9-nicotinic response in isolated saccular hair cells. These enzymes when inactivated became completely ineffective at abolishing the a9-nicotinic response, suggesting an enzymatic interaction with the alpha 9nAChR and/or downstream effector. The mechanism by which these enzymes render saccular hair cells unresponsive to acetylcholine remains unknown, but it most likely involves proteolysis of alpha 9nAChR, SK, or both. (C) 2001 Elsevier Science B.V. All rights reserved.