CAJ | 학술논문

目的用一种改良的灌流方法研究和再次证实HEK 293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,pH 5.0的细胞外液诱发的酸敏感性1a型离子通道电流足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HEK 293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.
목적 용일충개량적관류방법연구화재차증실HEK 293세포내원성산민감성리자통도적생물물리화약이학특성.방법 사용전세포막편겸기술,장기록세포여파편분리,병사세포위우수액관전,정표부상태,이기록재저pH치외액중,HEK293세포적산민감성1a형리자통도적전류.결과 사용세포표부방법,pH 5.0적세포외액유발적산민감성1a형리자통도전류족전통세포부착법유발적량배.재량충불동적방법하,통도전류체도봉치적시간분별시(21±5)ms화(270±25)ms,실활적시간상수분별시(496±23)ms화(2284±120)ms.세포표류법야명현증강amiloride대산민감성1a형리자통도적조단효능.량충방법구유상사적pH격활적EC50(분별위6.6±0.6,6.6±0.7).결론 산민감성1a형리자통도적격활수요세포외액쾌속적교환.통과세포표부적방법,아문재차증명료산민감성1a형리자통도전류적존재,경정학지분석료HEK 293세포내원성산민감형리자통도적생물물리화약이학특성.저일개량적방법능구용우연구소유적산민감형리자통도화수요쾌속세포외액교환적배체문공통도.
Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel la (hASICla) 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 hASIC1a [IC50 is (3.4±1.1) μmol/L and (2.4±0.9) μmol/L,respectively].Both recording methods have similar pH activation EC50 (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 hASICla current was re-confirmed and the biophysical and pharmacological properties of hASIC1a 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.