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.
Correia
Manning J
MJ
Department of Otolaryngology, University of Texas Medical Branch, Galveston, Texas 77555-1063, USA. mjcorrei@utmb.edu
Wood
Thomas G
TG
Prusak
Deborah
D
Weng
Tianxiang
T
Rennie
Katherine J
KJ
Wang
Hui-Qun
HQ
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D010856:Columbidae / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D004594:Electrophysiology; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D018069:Hair Cells, Vestibular / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics; D018408:Patch-Clamp Techniques / Q000379:methods; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000235:genetics*; D012313:RNA / Q000235:genetics
D030161:Avian Proteins; D024661:Potassium Channels, Inwardly Rectifying; D012313:RNA