Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function
Axel R. Concepcion, Martin Vaeth, Larry E. Wagner II, Miriam Eckstein, Lee Hecht, Jun Yang, David Crottes, Maximilian Seidl, Hyosup P. Shin, Carl Weidinger, Scott Cameron, Stuart E. Turvey, Thomas Issekutz, Isabelle Meyts, Rodrigo S. Lacruz, Mario Cuk, David I. Yule, Stefan Feske
Axel R. Concepcion, Martin Vaeth, Larry E. Wagner II, Miriam Eckstein, Lee Hecht, Jun Yang, David Crottes, Maximilian Seidl, Hyosup P. Shin, Carl Weidinger, Scott Cameron, Stuart E. Turvey, Thomas Issekutz, Isabelle Meyts, Rodrigo S. Lacruz, Mario Cuk, David I. Yule, Stefan Feske
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Research Article Cell biology Dermatology

Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function

Abstract

Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release–activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel–deficient patients and mice with ectodermal tissue–specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

Authors

Axel R. Concepcion, Martin Vaeth, Larry E. Wagner II, Miriam Eckstein, Lee Hecht, Jun Yang, David Crottes, Maximilian Seidl, Hyosup P. Shin, Carl Weidinger, Scott Cameron, Stuart E. Turvey, Thomas Issekutz, Isabelle Meyts, Rodrigo S. Lacruz, Mario Cuk, David I. Yule, Stefan Feske

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Figure 8

SOCE-induced Cl– currents in human sweat gland cells are mediated by TMEM16A.

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SOCE-induced Cl– currents in human sweat gland cells are mediated by TME...
(A–E) NCL-SG3 cells were stably transduced with shBEST2 and shTMEM16A, and analyzed for Cl– currents by patch clamping in whole-cell configuration. (A) Recording protocol (top): Currents were elicited by 20-mV steps from –80 mV to 120 mV (from a holding potential of –50 mV) followed by a 0.5-second hyperpolarizing step to –80 mV. Representative current traces (bottom) recorded in individual shBEST2- and shTMEM16A-transduced NCL-SG3 cells with 1 μM Ca2+ present in the patch pipette. (B) Current density versus voltage relationships determined at the end of each test pulse from experiments shown in A. Data are mean ± SEM of 5 cells for shBEST2 and 8 cells for shTMEM16A. (C) Ca2+ influx in shRNA-transduced NCL-SG3 cells following agonist stimulation was measured and analyzed as described in Figure 6F. Representative [Ca2+]i traces from 1 experiment (left) and quantitation of peak [Ca2+]i at 60 seconds and 300 seconds, and the overall [Ca2+]i increase (AUC) from 50–300 seconds. Data are mean ± SEM of 4 independent experiments with at least 10 cells analyzed per experiment. (D) Current densities in NCL-SG3 cells stimulated with 10 μM trypsin in 2 mM Ca2+-containing extracellular buffer. Currents were elicited by consecutive 2-second voltage steps to 80 mV, followed by a 0.5-second hyperpolarizing step to –80 mV. Data are mean ± SEM of 8 cells for shBEST2 and 11 cells for shTMEM16A. (E) Representative Cl– current traces from individual shBEST2- and shTMEM16A-transduced NCL-SG3 cells extracted at 0 seconds, 60 seconds, and 300 seconds from the experiment shown in D. (F) Schematic representation of SOCE-induced TMEM16A activation and Cl– secretion. AChR stimulation results in IP3-mediated release of Ca2+ from ER stores, which activates STIM1 and STIM2, allowing them to bind to and open the store-operated CRAC channel ORAI1. SOCE via ORAI1 activates TMEM16A Cl– channels and Cl– secretion, which provides the osmotic driving force for water flow and sweat production. For details see text. TEA-Cl, tetraethylammonium chloride.