Hayashi M, Komazaki S, Ishikawa T
Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkido University, Sapporo 060-0818, Japan.
J Physiol. 2003 Feb 15;547(Pt 1):255-69. doi: 10.1113/jphysiol.2002.035857. Epub 2003 Jan 3.
Using electrophysiological and molecular techniques, we investigated the molecular nature of an inwardly rectifying K+ channel in bovine parotid acinar (BPA) cells and examined its role in setting resting membrane potential. In whole-cell recordings from freshly isolated BPA cells, a predominant current was a K+ current rectified strongly in the inward direction. An inward conductance of the inwardly rectifying K+ (Kir) current was proportional to [K+]o(0.57). The selectivity sequence based on permeability ratios was K+ (1.00) > Rb+ (0.63) >> Li+ (0.04) = Na+ (0.02) and the sequence based on conductance ratios was K+ (1.00) >> Rb+ (0.03) = Li+ (0.03) = Na+ (0.02). The current was blocked by extracellular Ba2+ and Cs+ in a voltage- and a concentration-dependent manner, with a Kd at 0 mV of 11.6 microM and 121 mM, respectively. Cell-attached patch measurements identified 27 pS K+ channels as being the most likely to mediate whole-cell Kir currents. Addition of Ba2+ (100 microM) to the bathing solution reversibly depolarized the resting membrane potential in intact unstimulated cells. RT-PCR of RNA from bovine parotid cells revealed transcripts of bovine Kir2.1 (bKir2.1). HEK293 cells stably expressing bKir2.1 cloned from bovine parotid exhibited whole-cell and single channel Kir currents, of which electrophysiological characteristics were quantitatively similar to those of native Kir currents. Immunohistochemical studies showed a bKir2.1 immunoreactivity in BPA cells. Collectively, these results suggest that Kir2.1 may mediate native Kir currents responsible for setting resting membrane potential in BPA cells and might be, at least in part, involved in spontaneous secretion in ruminant parotid glands.
我们运用电生理和分子技术，研究了牛腮腺腺泡（BPA）细胞内向整流钾通道的分子特性，并检测了其在设定静息膜电位中的作用。在新鲜分离的BPA细胞的全细胞记录中，一种主要电流是内向整流的钾电流。内向整流钾（Kir）电流的内向电导与[K⁺]ₒ（0.57）成正比。基于通透率的选择性顺序为K⁺（1.00）＞Rb⁺（0.63）＞＞Li⁺（0.04） = Na⁺（0.02），基于电导率的顺序为K⁺（1.00）＞＞Rb⁺（0.03） = Li⁺（0.03） = Na⁺（0.02）。该电流被细胞外Ba²⁺和Cs⁺以电压和浓度依赖的方式阻断，在0 mV时的Kd分别为11.6 μM和121 mM。细胞贴附式膜片钳测量确定27 pS钾通道最有可能介导全细胞Kir电流。向浴液中添加Ba²⁺（100 μM）可使完整未刺激细胞的静息膜电位可逆性去极化。对牛腮腺细胞RNA进行的逆转录聚合酶链反应（RT-PCR）显示了牛Kir2.1（bKir2.1）的转录本。稳定表达从牛腮腺克隆的bKir2.1的人胚肾293（HEK293）细胞表现出全细胞和单通道Kir电流，其电生理特性在数量上与天然Kir电流相似。免疫组织化学研究显示BPA细胞中有bKir2.1免疫反应性。总体而言，这些结果表明Kir2.1可能介导负责设定BPA细胞静息膜电位的天然Kir电流，并且可能至少部分参与反刍动物腮腺的自发分泌。
