PG-00096-2004.R1 Molecular Characterization of an Inward Rectifier Channel (IKir) found in Avian Vestibular Hair Cells: Cloning and Expression of pKir 2.1

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 Kir 2.0 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 Kir 2.1 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 1284 base pair nucleotide sequence, showed 94, 85 and 83 % identity with Kir 2.1 subunit sequences from chick lens, and Kir 2 sequences from human heart, and a mouse macrophage cell line, respectively. Phylogenetic analyses revealed that pKir 2.1 formed an immediate node with hKir2.1 but not with hKir 2.2 2.4. Hair cells (type I and type II) and supporting cells in the sensory epithelium reacted positively with a Kir 2.1 antibody. The whole cell current recorded in oocytes and CHO cells, transfected with pigeon hair cell Kir 2.1 (pKir 2.1.), demonstrated blockage by Ba and sensitivity to changing K concentration. The mean single channel linear slope conductance in transfected CHO cells was 29pS. The open dwell time was long (~300ms at -100mV) and the closed dwell time was short (~34ms at -100 mV). Multi-states ranging from 3-6 were noted in some single channel responses. All of the above features have been described for other Kir 2.1 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 Ba depolarized the resting membrane potential by ~30 mV. Negative currents hyperpolarized the membrane ~20mV before block but ~60 mV following block. RT-PCR studies revealed that the pKir 2.1 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.