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

Tembo, Maiwase; Wozniak, Katherine L.; Bainbridge, Rachel E.; Carlson, Anne E.

doi:10.1074/jbc.ra118.007128

Cited by 46 publications

(61 citation statements)

References 53 publications

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“…Thus, the search for activators or potentiators of TMEM16A should also consider the activatory pathway described for CLCA1. and overexpressed TMEM16A [18][19][20]34]. In contrast to the poorly understood mechanisms of the currently available activators of TMEM16A, such as Eact, cinnamaldehyde, ETX001, and others, the mechanism by which the TMEM16A-activator CLCA-1 increases membrane expression of TMEM16A has been described earlier [35].…”

Section: Compoundmentioning

confidence: 99%

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Centeio

Cabrita

Benedetto

et al. 2020

IJMS

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TMEM16A is a Ca 2+ activated Cl − channel with important functions in airways, intestine, and other epithelial organs. Activation of TMEM16A is proposed as a therapy in cystic fibrosis (CF) to reinstall airway Cl − secretion and to enhance airway surface liquid (ASL). This CFTR-agnostic approach is thought to improve mucociliary clearance and lung function in CF. This could indeed improve ASL, however, mucus release and airway contraction may also be induced by activators of TMEM16A, particularly in inflamed airways of patients with asthma, COPD, or CF. Currently, both activators and inhibitors of TMEM16A are developed and examined in different types of tissues. Here we compare activation and inhibition of endogenous and overexpressed TMEM16A and analyze potential off-target effects. The three well-known blockers benzbromarone, niclosamide, and Ani9 inhibited both TMEM16A and ATP-induced Ca 2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca 2+ activated TMEM16A, also induced a subtle but significant Ca 2+ store release and inhibited store-operated Ca 2+ influx. Niclosamide, benzbromarone and Ani9 also affected TMEM16F whole cell currents, indicating limited specificity for these inhibitors. The compounds Eact, cinnamaldehyde, and melittin, as well as the phosphatidylinositol diC8-PIP 2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT 29 colonic epithelial cells. In contrast, TMEM16A overexpressed in HEK293 cells was potently stimulated by these activators. We speculate that overexpressed TMEM16A might have a better accessibility to intracellular Ca 2+ , which causes spontaneous activity even at basal intracellular Ca 2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A.

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Section: Compoundmentioning

confidence: 99%

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Centeio

Cabrita

Benedetto

et al. 2020

IJMS

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“…Phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) was recently shown to plays a critical role in regulating TMEM16A and TMEM16F ion channel rundown or desensitization (Ta et al ., 2017; De Jesús-Pérez et al ., 2018; Ye et al ., 2018; S. C. Le et al ., 2019; Tembo et al ., 2019; Yu et al ., 2019). In addition, both intracellular and extracellular pH have also been reported to regulate endogenous CaCCs (Arreola, Melvin and Begenisich, 1995; Park and Brown, 1995; Qu and Hartzell, 2000) and heterologous expressed TMEM16A CaCCs (Chun et al ., 2015; Cruz-Rangel et al ., 2017; Segura-Covarrubias et al ., 2020).…”

Section: Introductionmentioning

confidence: 99%

Molecular underpinning of intracellular pH regulation on TMEM16F

Liang¹,

Yang

2020

Preprint

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39TMEM16F, a dual functional phospholipid scramblase and ion channel, is important in blood 40 coagulation, skeleton development, HIV infection and cell fusion. Despite the advances in 41 understanding its structure and activation mechanism, how TMEM16F is regulated by intracellular 42 factors remains largely elusive. Here we report that TMEM16F lipid scrambling and ion channel 43 activities are strongly influenced by intracellular pH (pHi). We find that low pHi attenuates 44 whereas high pHi potentiates TMEM16F activation. Our biophysical characterizations pinpoint 45 that the pHi regulatory effects on TMEM16F stem from protonation and deprotonation of the Ca 2+ 46 binding sites, which in turn reduces and enhances Ca 2+ binding affinity, respectively. We further 47 demonstrate that intracellular alkalization of 0.5 pHi can significantly promote TMEM16F 48 activities in a choriocarcinoma cell line. Our findings thus uncover a regulatory mechanism of 49 TMEM16F by pHi and shine light on understanding the pathophysiological roles of TMEM16F in 50 diseases with dysregulated pHi including cancer. 51 52

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“…23,26 Both PIP2 and Ca 2+ are required for sustained activation of TMEM16A CaCC. 24 Simulations of TMEM16A in the Ca 2+ -free state with PIP2 docked to the specific binding pocket suggest that PIP2 can maintain stable contact with the same set of basic residues, R455, R486, K571 and R579, with contact probabilities of 0.24, 0.50, 0.96 and 0.99, respectively. However, the pore remained collapsed in all three simulations (Fig.…”

Section: Discussionmentioning

confidence: 94%

“…6,34 The opening transitions appear to be transient and showing that PIP2 is required for maintaining the conductive state of TMEM16A even under saturating Ca 2+ concentrations. 24,25,29 TMs 3-6 are colored in red, TM4 in blue and the rest of the protein in cyan. Two of the inner gate residues (L547 and I641) and key pore-lining residues above the gate (V543, I640 and P595) are represented as yellow and blue sticks, respectively.…”

Section: Specific Pip2 Binding To Ca 2+ -Bound Tmem16a Induces Spontamentioning

confidence: 99%

“…Besides Ca 2+ signaling and membrane potential, the channel activity of TMEM16A is also regulated by phosphatidylinositol (4,5)-bisphosphate (PIP2). 23,24,25,26,27 Upon prolonged Ca 2+ activation, TMEM16A CaCCs undergo time-dependent current decay even under saturating Ca 2+ concentrations (> 1 µM). 25,28 Such desensitization or rundown after activation can be inhibited and reversed by binding of PIP2.…”

Section: Introductionmentioning

confidence: 99%

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Specific PIP₂Binding Promotes Calcium Activation of TMEM16A Chloride Channels

Jia

Chen

2020

Preprint

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TMEM16A is a widely expressed Ca 2+ -activated Clchannel that regulates crucial physiological functions including fluid secretion, neuronal excitability, and smooth muscle contraction. There is a critical need to understand the molecular mechanisms of TMEM16A gating and regulation.However, high-resolution TMEM16A structures have failed to reveal an activated state with unobstructed permeation pathway even with saturating Ca 2+ . This has been attributed to the requirement of PIP2 for preventing TMEM16A desensitization. Here, we show that specific binding PIP2 to TMEM16A can lead to spontaneous opening of the permeation pathway in the Ca 2+ -bound state. The predicted activated state is highly consistent with a wide range of mutagenesis and functional data. It yields a maximal Clconductance of ~1 pS, similar to experimental estimates, and recapitulates the selectivity of larger SCNover Cl -. The resulting molecular mechanism of activation provides a basis for understanding the interplay of multiple signals in controlling TMEM16A channel function.

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

Cited by 46 publications

References 53 publications

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Molecular underpinning of intracellular pH regulation on TMEM16F

Specific PIP₂Binding Promotes Calcium Activation of TMEM16A Chloride Channels

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

Cited by 46 publications

References 53 publications

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Molecular underpinning of intracellular pH regulation on TMEM16F

Specific PIP2Binding Promotes Calcium Activation of TMEM16A Chloride Channels

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Specific PIP₂Binding Promotes Calcium Activation of TMEM16A Chloride Channels