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The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop

The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified... Ca 2+ -activated Cl − channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure–function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca 2+ dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca 2+ concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates 386 EEEEE 390 and 399 EYE 401 . The EYE deletion did not significantly modify the apparent Ca 2+ dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca 2+ affinity but modified the voltage dependence, shifting the conductance–voltage relations toward more positive voltages. These findings indicate that glutamates E367 and 386 EEEEE 390 in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure–function study for this channel. Submitted: 27 December 2011 Accepted: 17 February 2012 This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms ). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ ). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of General Physiology Rockefeller University Press

The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop

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Publisher
Rockefeller University Press
Copyright
© 2012 Cenedese et al.
ISSN
0022-1295
eISSN
1540-7748
DOI
10.1085/jgp.201110764
pmid
22412191
Publisher site
See Article on Publisher Site

Abstract

Ca 2+ -activated Cl − channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure–function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca 2+ dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca 2+ concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates 386 EEEEE 390 and 399 EYE 401 . The EYE deletion did not significantly modify the apparent Ca 2+ dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca 2+ affinity but modified the voltage dependence, shifting the conductance–voltage relations toward more positive voltages. These findings indicate that glutamates E367 and 386 EEEEE 390 in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure–function study for this channel. Submitted: 27 December 2011 Accepted: 17 February 2012 This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms ). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ ).

Journal

The Journal of General Physiology – Rockefeller University Press

Published: Apr 1, 2012

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