A role for chloride in the hyperpolarizing effect of acetylcholine in isolated frog vestibular hair cells.

Acetylcholine (ACh) is the dominant transmitter released from inner ear efferent neurons. In frog vestibular organs, these efferent neurons synapse exclusively with type II hair cells. Hair cells isolated from the frog saccule hyperpolarize following the application of 50 microM ACh, thereby demonstrating the presence of an ACh receptor. A role for Cl(-) in the response of hair cell-bearing organs to efferent nerve activation or ACh application was suggested some years ago. Perfusion with solutions in which most of the Cl(-) was replaced by large impermeant anions decreased the cholinergic inhibition of afferent firing in the cat and turtle cochleas, and frog semicircular canal. Our previous work in the intact organ demonstrated that substitution of large impermeant anions for Cl(-) or use of Cl(-) channel blockers reduced the effect of ACh on saccular afferent firing. Using the perforated-patch clamping technique, replacement of Cl(-) by methanesulfonate, iodide, nitrate, or thiocyanate attenuated the hyperpolarizing response to ACh in hair cells isolated from the frog saccule. The chloride channel blockers picrotoxin and 4,4'-dinitrostilbene-2,2'-disulfonic acid were also tested and found to inhibit the ACh response. Thus, the present work demonstrates that the effects of Cl(-) substitutions or Cl(-) channel blockers on the ACh response in the intact saccule can be explained completely by effects on the hair cell. Evidence is also presented for the presence of the messenger RNA for a calcium-dependent chloride channel in all hair cells but especially saccular hair cells. This channel may be involved in the response to ACh. The precise role for chloride in this response, whether as a distinct ion current, as a transported ion, or as a permissive ion for other components, is discussed.