Electrophysiological characterization of native Na+–HCO3– cotransporter current in bovine parotid acinar cells
Electrophysiological characterization of native Na+–HCO3– cotransporter current in bovine parotid acinar cells
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DOI：
10.1113/jphysiol.2005.088633
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发表时间：
2005-10
期刊：
影响因子：
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通讯作者：
S. Yamaguchi;T. Ishikawa
中科院分区：
文献类型：
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作者：
S. Yamaguchi;T. Ishikawa
使用膜片钳和分子生物学技术，我们确定并表征了急性分离的牛腮腺腺泡（BPA）细胞中最可能由产电Na+-HCO 3 −协同转运蛋白（NBCe）产生的膜电流。当BPA细胞用富含N-甲基-d-葡糖胺（NMDG）-谷氨酸盐的移液器溶液透析时，将富含谷氨酸钠、名义上不含HCO 3 −的浴槽溶液转换为含有25 mm HCO 3 −但不含Cl−的浴槽溶液，引起具有线性电流-电压关系的全细胞电流。用NMDG或0.5 mm的4，4 ′-二异硫氰基-二苯乙烯-2，2 ′-二磺酸（DIDS）完全替代细胞外Na+（Na+o）可消除HCO 3 −诱发的电流，并且仅部分得到Li+o的支持，但不受K+o、Cs+o和胆碱的支持。由[Na+]o变化引起的DIDS（0.5 mm）敏感电流的反向电位偏移对应于HCO 3 −与Na+的表观耦合比为2。RT-PCR分析显示BPA细胞中存在NBCe 1-B的转录物，但不存在NBCe 1-A。从转染NBCe 1-B（从BPA细胞克隆）的HEK 293细胞中记录的全细胞电流的电生理学和药理学特性与天然电流相似。在细胞贴附贴片配置中膜电位变化的非侵入性测量表明，NBCe活性存在于浸泡在含25 mm HCO 3 −溶液中的完整未刺激BPA细胞中。总的来说，这些结果不仅表明NBCe存在，功能性，并可能介导，至少部分地，由NBCe 1-B在BPA细胞，但也提供了第一个电生理特性的运输特性的NBCe表达在天然外分泌腺。
Using patch‐clamp and molecular biological techniques, we identified and characterized membrane currents most likely generated by an electrogenic Na+–HCO3− cotransporter (NBCe) in acutely dissociated bovine parotid acinar (BPA) cells. When BPA cells were dialysed with a N‐methyl‐d‐glucamine (NMDG)‐glutamate‐rich pipette solution, switching a Na‐glutamate‐rich, nominally HCO3−‐free bath solution to the one containing 25 mm HCO3−, but not Cl−, elicited a whole‐cell current with a linear current–voltage relation. The HCO3− evoked current was abolished by total replacement of extracellular Na+ (Na+o) with NMDG or by 0.5 mm 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonic acid (DIDS), and was only partially supported by Li+o, but not by K+o, Cs+o, and cholineo. The reversal potential shift of DIDS (0.5 mm)‐sensitive current induced by a change of [Na+]o corresponded to an apparent coupling ratio of HCO3− to Na+ of 2. RT‐PCR analysis showed the presence of transcripts of NBCe1‐B, but not NBCe1‐A in BPA cells. Electrophysiological and pharmacological properties of whole‐cell currents recorded from HEK293 cells transfected with the NBCe1‐B, which was cloned from BPA cells resembled those of the native currents. Non‐invasive measurements of membrane potential changes in the cell‐attached patch configuration indicated that an NBCe activity is present in intact unstimulated BPA cells bathed in a 25 mm HCO3−‐containing solution. Collectively, these results not only suggest that an NBCe is present, functional and may be mediated, at least in part, by NBCe1‐B in BPA cells, but also provide the first electrophysiological characterization of transport properties of NBCe expressed in native exocrine glands.