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

Haiyue Lin,Joo Han Woo,Joo Hyun Nam,Sung Joon Kim,Ikhyun Jun,Min Goo Lee

doi:10.1038/s41598-019-43162-1

Haiyue Lin, Joo Han Woo + Show 4 more

Open Access

https://doi.org/10.1038/s41598-019-43162-1

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Abstract

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 time 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.

Highlights

The TMEM16 family of proteins, known as anoctamins (ANOs), comprise several members that function as calcium (Ca2+)-activated chloride channels (CaCCs)[1]
To confirm ANO6 variant functional activity, a whole-cell patch clamp assay was conducted for mock (green fluorescent protein (GFP)), variant type 1 (V1), V2, V3, and V5 of ANO6-transfected HEK293T cells using symmetrical N-methyl-D-glucamine-Cl (NMDG-Cl) solutions with 100 μM [Ca2+]i, which is known as the maximal Ca2+ concentration for ANO6 variant activation[28]
Despite the expression of ANO6 (TMEM16F) in various cell types[26,32], the physiological roles of ANO6 have been identified in only limited types of cells such as immunocytes, platelets, and osteoblasts[9,13,14,15], all of which are thought to be related to its PS scramblase activity[33,34,35]

Summary

Introduction

The TMEM16 family of proteins, known as anoctamins (ANOs), comprise several members that function as calcium (Ca2+)-activated chloride channels (CaCCs)[1]. The role of ANO6 as a physiologically meaningful ion channel remains controversial This debate largely stems from the fact that ANO6 activation requires a very high intracellular Ca2+ concentration ([Ca2+]i). Under the whole-cell patch clamp, a ramp-like pulse from −100 mV to 100 mV (duration time 3 s) was applied every 20 s (holding voltage, −60 mV) and the step pulse protocol was started from a holding potential of −60 mV and depolarised for 0.5 s from −100 mV to +100 mV with 20 mV increments. (f) Summary bar graph of delay time (t1/2, peak) of IANO6 generation (time from the start of whole-cell patch recordings to half maximal current activation) detected with 100 μM [Ca2+]i. These properties are largely different from those of the ANO1 current that is activated with a submicromolar EC50 (~400 nM) of [Ca2+]i, showing no delayed activation in response to Ca2+ signals[23,24]. Because of the relatively low Ca2+ sensitivity and delayed activation properties of ANO6, unlike ANO1, which is activated directly by ER calcium release during G protein coupled receptor (GPCR) stimulation, ANO6 might be activated by sustained calcium influx through store-operated calcium entry after GPCR stimulation[25]

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