Correia Manning J, Wood Thomas G, Prusak Deborah, Weng Tianxiang, Rennie Katherine J, Wang Hui-Qun
Department of Otolaryngology, University of Texas Medical Branch, Galveston, Texas 77555-1063, USA.
Physiol Genomics. 2004 Oct 4;19(2):155-69. doi: 10.1152/physiolgenomics.00096.2004. Epub 2004 Aug 17.
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.
在几种物种的内耳部分感觉细胞（毛细胞）中观察到一种快速内向整流电流。虽然该电流被推测为IKir电流，但关于克隆通道是否真的属于钾内向整流器的Kir2.0亚家族存在相互矛盾的证据。在本文中，我们首次报告了来自电生理、生化、免疫组织化学和遗传学研究的趋同证据，这些证据表明Kir2.1通道携带了在鸽前庭毛细胞中发现的快速内向整流电流。从单个II型和多个鸽前庭毛细胞中提取细胞质后，对mRNA进行逆转录、扩增和测序。由1284个碱基对的核苷酸序列组成的开放阅读框（ORF），与鸡晶状体的Kir2.1亚基序列、人心的Kir2序列和小鼠巨噬细胞系的序列分别具有94%、85%和83%的同一性。系统发育分析表明，pKir2.1与hKir2.1形成一个直接节点，而与hKir2.2 - 2.4不形成节点。感觉上皮中的毛细胞（I型和II型）和支持细胞与Kir2.1抗体发生阳性反应。用鸽毛细胞Kir2.1（pKir2.1）转染的卵母细胞和CHO细胞中记录的全细胞电流，表现出Ba2+阻断以及对K+浓度变化的敏感性。转染的CHO细胞中的平均单通道线性斜率电导为29 pS。开放驻留时间长（在 - 100 mV时约为300 ms），关闭驻留时间短（在 - 100 mV时约为34 ms）。在一些单通道反应中观察到3 - 6个多状态。上述所有特征在其他Kir2.1通道中也有描述。对天然鸽前庭毛细胞的电流钳研究说明了该通道可能的生理作用，并表明Ba2+对通道的阻断使静息膜电位去极化约为30 mV。负电流在阻断前使膜超极化约20 mV，但在阻断后使膜超极化约60 mV。RT-PCR研究表明，在鸽前庭毛细胞中发现的pKir2.1通道也存在于鸽前庭神经、前庭神经节、晶状体、颈部肌肉、脑（脑干、小脑和视顶盖）、肝脏和心脏中。
