Responses of pigeon vestibular hair cells to cholinergic agonists and antagonists.

Acetylcholine (ACh) is the major neurotransmitter released from vestibular efferent terminals onto hair cells and afferents. Previous studies indicate that the two classes of acetylcholine receptors, nicotinic (nAChRs) and muscarinic receptors (mAChRs), are expressed by vestibular hair cells (VHCs). To identify if both classes of receptors are present in VHCs, whole cell, voltage-clamp- and current-clamp-patch recordings were performed on isolated pigeon vestibular type I and type II HCs during the application of the cholinergic agonists, acetylcholine and carbachol, and the cholinergic antagonists, D-tubocurarine and atropine. By applying in different combinations, these compounds were used to selectively activate either nAChRs or mAChRs. The effects of nAChR and mAChR activation on HC currents and zero electrode current potential (V(z)) were monitored. It was found that presumed mAChR activation decreased both inward and outward currents in both type I and type II HCs, resulting in either a depolarization or hyperpolarization. Conversely, nAChR activation mainly increased both inward and outward currents in type II HCs, resulting in a hyperpolarization of their V(z). nAChR activation also increased outward currents in type I HCs resulting in either a depolarization or hyperpolarization of their V(z). The decrease of inward and outward currents and the depolarization of the V(z) in type I pigeon HCs by activation of mAChRs represents a new finding. Ion channel candidates in pigeon vestibular HCs that might underlie the modulation of the macroscopic ionic currents and V(z) by different AChR activation are discussed.

Molecular characterization of an inward rectifier channel (IKir) found in avian vestibular hair cells: cloning and expression of pKir2.1.

A fast inwardly rectifying current has been observed in some of the sensory cells (hair cells) of the inner ear of several species. While the current was presumed to be an IKir current, contradictory evidence existed as to whether the cloned channel actually belonged to the Kir2.0 subfamily of potassium inward rectifiers. In this paper, we report for the first time converging evidence from electrophysiological, biochemical, immunohistochemical, and genetic studies that show that the Kir2.1 channel carries the fast inwardly rectifying currents found in pigeon vestibular hair cells. Following cytoplasm extraction from single type II and multiple pigeon vestibular hair cells, mRNA was reverse transcribed, amplified, and sequenced. The open reading frame (ORF), consisting of a 1,284-bp nucleotide sequence, showed 94, 85, and 83% identity with Kir2.1 subunit sequences from chick lens, Kir2 sequences from human heart, and a mouse macrophage cell line, respectively. Phylogenetic analyses revealed that pKir2.1 formed an immediate node with hKir2.1 but not with hKir2.2-2.4. Hair cells (type I and type II) and supporting cells in the sensory epithelium reacted positively with a Kir2.1 antibody. The whole cell current recorded in oocytes and CHO cells, transfected with pigeon hair cell Kir2.1 (pKir2.1), demonstrated blockage by Ba2+ and sensitivity to changing K+ concentration. The mean single-channel linear slope conductance in transfected CHO cells was 29 pS. The open dwell time was long (approximately 300 ms at -100 mV), and the closed dwell time was short (approximately 34 ms at -100 mV). Multistates ranging from 3-6 were noted in some single-channel responses. All of the above features have been described for other Kir2.1 channels. Current clamp studies of native pigeon vestibular hair cells illustrated possible physiological roles of the channel and showed that blockage of the channel by Ba2+ depolarized the resting membrane potential by approximately 30 mV. Negative currents hyperpolarized the membrane approximately 20 mV before block but approximately 60 mV following block. RT-PCR studies revealed that the pKir2.1 channels found in pigeon vestibular hair cells were also present in pigeon vestibular nerve, vestibular ganglion, lens, neck muscle, brain (brain stem, cerebellum and optic tectum), liver, and heart.