Abstract：
Selective calcium chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N\',N\'-tetraacetic acid (BAPTA) is a common tool to investigate calcium signaling. However, BAPTA expresses various effects on intracellular calcium signaling, which are not related to its ability to bind Ca2+. In patch clamp experiments, we investigated calcium chelation independent effects of BAPTA on endogenous calcium-activated chloride channels ANO6 (TMEM16F) in HEK293T cells. We have found that application of BAPTA to intracellular solution led to two distinct effects on channels properties. On the one hand, application of BAPTA acutely reduced amplitude of endogenous ANO6 channels induced by 10 μM Ca2+ in single channel recordings. On the other hand, BAPTA application by itself induced ANO6 channel activity in the absence of the intracellular calcium elevation. Open channel probability was enhanced by increasing the intracellular BAPTA concentration from 0.1 to 1 and 10 mM. Another calcium chelator EGTA did not demonstrate chelation independent effects on the ANO6 activity in the same conditions. Due to off-target effects BAPTA should be used with caution when studying calcium-activated ANO6 channels.
摘要：
选择性钙螯合剂1,2-二(2-氨基苯氧基)乙烷-N,N,N\',N'-四乙酸（BAPTA）是研究钙信号的常用工具。然而,BAPTA对细胞内钙信号表达各种作用，这与它结合Ca2+的能力无关。在膜片钳实验中,我们研究了BAPTA对HEK293T细胞内源性钙激活氯通道ANO6(TMEM16F)的钙螯合非依赖性作用。我们已经发现,将BAPTA应用于胞内溶液导致对通道性质的两种不同影响。一方面,BAPTA的应用在单通道记录中急剧降低了10μMCa2诱导的内源性ANO6通道的振幅。另一方面,在没有细胞内钙升高的情况下，BAPTA的应用本身会诱导ANO6通道活性。通过将细胞内BAPTA浓度从0.1增加到1和10mM，可以提高开放通道的可能性。另一种钙螯合剂EGTA在相同条件下对ANO6活性没有显示出螯合非依赖性作用。由于脱靶效应，在研究钙激活的ANO6通道时，应谨慎使用BAPTA。
