Temperature-dependent increase in the calcium sensitivity and acceleration of activation of ANO6 chloride channel variants.

Anoctamin-6 (ANO6) belongs to a family of calcium (Ca²⁺)-activated chloride channels (CaCCs), with three splicing variants (V1, V2, and V5) showing plasma membrane expression. Unlike other CaCCs, ANO6 requires a non-physiological intracellular free calcium concentration ([Ca²⁺]_i > 1 μM) and several minutes for full activation under a whole-cell patch clamp. Therefore, its physiological role as an ion channel is uncertain and it is more commonly considered a Ca²⁺-dependent phospholipid scramblase. Here, we demonstrate that physiological temperature (37 °C) increases ANO6 Ca²⁺ sensitivity under a whole-cell patch clamp; V1 was activated by 1 μM [Ca²⁺]_i, whereas V2 and V5 were activated by 300 nM [Ca²⁺]_i. Increasing the temperature to 42 °C led to activation of all ANO6 variants by 100 nM [Ca²⁺]_i. The delay t_ime for activation of the three variants was significantly shortened at 37 °C. Notably, the temperature-dependent Ca²⁺-sensitisation of ANO6 became insignificant under inside-out patch clamp, suggesting critical roles of unknown cytosolic factors. Unlike channel activity, 27 °C but not 37 °C (physiological temperature) induced the scramblase activity of ANO6 at submicromolar [Ca²⁺]_i (300 nM), irrespective of variant type. Our results reveal a physiological ion conducting property of ANO6 at 37 °C and suggest that ANO6 channel function acts separately from its scramblase activity.

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