An optimized recording method to characterize biophysical and pharmacological properties of acid-sensing ion channel  

一种改良的精确分析酸敏感性离子通道特性的优化记录方法(英文)

作　　者：李爱[1] 司文[2] 胡新武[1] 刘长金[1] 曹晓华[2] 

机构地区：[1]华中科技大学同济医学院基础医学院生理系,武汉430030 [2]华东师范大学脑功能基因组学教育部重点实验室上海市科学技术委员会重点实验室,上海200062

出　　处：《Neuroscience Bulletin》2008年第3期160-165,共6页神经科学通报（英文版）

摘　　要：Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods. Methods With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASICla currents evoked by low pH external solution. Results Using cell floating method, the amplitude of hASICla currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is （21±5） ms and （270±25） ms, respectively. Inactivation time constants are （496±23） ms and （2284±120） ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC 1 a [IC50 is （3.4± 1.1 ） μmol/L and （2.4± 0.9） μmol/L, respectively]. Both recording methods have similar pH activation ECs0 （6.6±0.6, 6.6±0.7, respectively）. Conclusion ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC la current was re-confirmed and the biophysical and pharmacological properties of hASIC la channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.目的用一种改良的灌流方法研究和再次证实HEK293细胞内源性酸敏感性离子通道的生物物理和药理学特性。方法使用全细胞膜片钳技术,将记录细胞与玻片分离,并使细胞位于输液管前,呈漂浮状态,以记录在低pH值外液中,HEK293细胞的酸敏感性1a型离子通道的电流。结果使用细胞漂浮方法,pH5.0的细胞外液诱发的酸敏感性1a型离子通道电流是传统细胞附着法诱发的两倍。在两种不同的方法下,通道电流达到峰值的时间分别是(21±5)ms和(270±25)ms,失活的时间常数分别是(496±23)ms和(2284±120)ms。细胞漂流法也明显增强amiloride对酸敏感性1a型离子通道的阻断效能。两种方法具有相似的pH激活的EC50(分别为6.6±0.6,6.6±0.7)。结论酸敏感性1a型离子通道的激活需要细胞外液快速的交换。通过细胞漂浮的方法,我们再次证明了酸敏感性1a型离子通道电流的存在,更精确地分析了HEK293细胞内源性酸敏感型离子通道的生物物理和药理学特性。这一改良的方法能够用于研究所有的酸敏感型离子通道和需要快速细胞外液交换的配体门控通道。

关 键 词：acid-sensing ion channel patch-clamp recording PH 

分 类 号：Q503[生物学—生物化学]