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Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ are both required to open the Cl- channel TMEM16A.

Authors
  • Tembo, Maiwase1
  • Wozniak, Katherine L1
  • Bainbridge, Rachel E1
  • Carlson, Anne E2
  • 1 Department of Biological Sciences, University of Pittsburgh, Pittsburgh Pennsylvania 15260.
  • 2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh Pennsylvania 15260 [email protected]
Type
Published Article
Journal
Journal of Biological Chemistry
Publisher
American Society for Biochemistry and Molecular Biology
Publication Date
Aug 16, 2019
Volume
294
Issue
33
Pages
12556–12564
Identifiers
DOI: 10.1074/jbc.RA118.007128
PMID: 31266809
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Transmembrane member 16A (TMEM16A) is a widely expressed Ca2+-activated Cl- channel with various physiological functions ranging from mucosal secretion to regulating smooth muscle contraction. Understanding how TMEM16A controls these physiological processes and how its dysregulation may cause disease requires a detailed understanding of how cellular processes and second messengers alter TMEM16A channel gating. Here we assessed the regulation of TMEM16A gating by recording Ca2+-evoked Cl- currents conducted by endogenous TMEM16A channels expressed in Xenopus laevis oocytes, using the inside-out configuration of the patch clamp technique. During continuous application of Ca2+, we found that TMEM16A-conducted currents decay shortly after patch excision. Such current rundown is common among channels regulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Thus, we sought to investigate a possible role of PIP2 in TMEM16A gating. Consistently, synthetic PIP2 rescued the current after rundown, and the application of PIP2 modulating agents altered the speed kinetics of TMEM16A current rundown. First, two PIP2 sequestering agents, neomycin and anti-PIP2, applied to the intracellular surface of excised patches sped up TMEM16A current rundown to nearly twice as fast. Conversely, rephosphorylation of phosphatidylinositol (PI) derivatives into PIP2 using Mg-ATP or inhibiting dephosphorylation of PIP2 using β-glycerophosphate slowed rundown by nearly 3-fold. Our results reveal that TMEM16A regulation is more complicated than it initially appeared; not only is Ca2+ necessary to signal TMEM16a opening, but PIP2 is also required. These findings improve our understanding of how the dysregulation of these pathways may lead to disease and suggest that targeting these pathways could have utility for potential therapies. © 2019 Tembo et al.

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