Calcium chelation independent effects of BAPTA on endogenous ANO6 channels in HEK293T cells

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.

Keywords: ANO6; BAPTA; Calcium-activated chloride channels; Ion channels; TMEM16F.