ANO1 Activation does not require Calmodulin

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The Ca2+-activated Cl channel anoctamin-1 (ANO1, also called TMEM16A), a member of the Anoctamin superfamily, plays a variety of important physiological roles including epithelial fluid secretion. ANO1 is activated and gated by intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to ANO1 or whether an additional Ca2+-binding subunit such as calmodulin (CaM) is required. Here, we report that CaM is not a necessary component for ANO1 activation for the following reasons: 1) ANO1 activation by photolysis of caged Ca2+ is very rapid (<1ms), suggesting that Ca2+ binds directly to the channel. 2) Exogenous CaM has no effect on the currents generated by either the ac or abc isoforms of ANO1 in inside-out excised patches. 3) Over-expression of functionally defective CaM has no effect on ANO1 (abc isoform) currents while it eliminates the small conductance Ca-activated K (SK2) current. 4) CaM does not physically interact with ANO1 as determined by co-immunoprecipitation. 5) ANO1 is activated in excised patches by Ba, which is a very poor CaM activator. We also re-examined the effect of the CaM inhibitor trifluoperazine (TFP). TFP blocks ANO1 in a voltage-dependent manner, suggesting that TFP blocks the current by lodging in the ANO1 pore. These results show CaM is not required for ANO1 activation and supports the hypothesis that Ca binds directly to ANO1.