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

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.

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

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 hASIC1a currents evoked by low pH external solution. RESULTS: Using cell floating method, the amplitude of hASIC1a 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) micromol/L and (2.4+/- 0.9) micromol/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 hASIC1a current was re-confirmed and the biophysical and pharmacological properties of hASIC1a channel in HEK293 cells were precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.