Chemically induced vesiculation as a platform for studying TMEM16F activity

Han, Tina W.; Ye, Wenlei; Bethel, Neville; Zubia, Mario; Kim, Andrew; Li, Kathy H.; Burlingame, Alma L.; Grabe, Michael; Jan, Yuh Nung

doi:10.1073/pnas.1817498116

Cited by 33 publications

(47 citation statements)

References 63 publications

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“…TMEM16F is also a Ca 2+ -activated small-conductance ion channel, yielding non-selective cation current in excised patch recordings (6,9). Consistently, activation of TMEM16F increases cytoplasmic Ca 2+ concentration in cells that heterologously express this channel (10). The intracellular Ca 2+ elevation might further activate TMEM16F, forming positive feedback that could potentially lead to detrimental consequences.…”

Section: Introductionmentioning

confidence: 81%

“…Transient transfection was performed with Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA, USA) 2 days before recording. The cDNA constructs for wild-type TMEM16F-mCherry, the mutants Q559K-, G615A-, G614A-, I612A-, G614_G615A-, N620A-, and E667Q-mCherry were stably transfected in HEK cells as previously reported (10). Briefly, the cDNAs were subcloned into pENTR1A (Addgene plasmid #17398) and transferred to pLenti CMV Hygro DEST (Addgene plasmid #17454) using Gateway cloning (24).…”

Section: Methodsmentioning

confidence: 99%

“…Similarly, alanine substitution of G615 of TMEM16F increased the Ca 2+ sensitivity (9, 10) and caused a left-shift of the voltage dependence recorded in 15 μM Ca 2+ ( Figure 3A, B). Notably, the glutamine at the lower segment of TM6 in TMEM16A, Q645, whose alanine substitution stabilizes the channel at a conformation similar to wild-type TMEM16A bound to one Ca 2+ ion (18), corresponds to a gap of the alignment in TMEM16F ( Alanine substitution of the asparagine "5-amino-acid-away" in TMEM16F TM6, N620, did not alter Ca 2+ sensitivity (10) (18). The comparison of gating between TMEM16A and TMEM16F is beyond the scope of this manuscript but will be briefly discussed later.…”

Section: Q559k Channel Ion Selectivity Varies With Intracellular Ca 2mentioning

confidence: 99%

“…In TMEM16A, Ca 2+ -binding may further change the electrostatic field of the permeation pathway and reduce the energy barrier that anions need to overcome to enter the pore (17). The possibility that charges in the binding pocket and in the pore can interact electrostatically, if applicable to TMEM16F, may provide an explanation for the observation that neutralization of the lysine in the permeation pathway of TMEM16F (K641A) increases the Ca 2+ sensitivity (10).…”

Section: Intracellular Ca 2+ and Membrane Depolarization Synergisticamentioning

confidence: 99%

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Dynamic Change of Electrostatic Field in TMEM16F Permeation Pathway Shifts Its Ion Selectivity

Ye¹,

Han²,

He³

et al. 2019

Preprint

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TMEM16F is activated by elevated intracellular Ca 2+ , and functions both as a small-conductance ion channel permeable to Ca 2+ and as a phospholipid scramblase. It is important to hold this positive feedback in check to prevent prolonged Ca 2+ -overloading in cells. We hypothesize that TMEM16F shifts its ion selectivity so that it is more permeable to Clthan cations at high intracellular Ca 2+ concentration. We tested this hypothesis with the Q559K mutant that shows no current rundown in excised patch with prolonged Ca 2+ elevation. Recorded in NaCl-based solution, the channel shifted its ion selectivity from Na + -selective to Cl --selective when intracellular Ca 2+ was increased. The ion selectivity switch did not correlate with changes of channel open state. Rather, it was indicative of an alteration of electrostatic field in the permeation pathway. Shifting ion-selectivity synergistically by intracellular divalent ions and membrane potential could work as a built-in mechanism that allows TMEM16F to brake the positive feedback.

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Section: Introductionmentioning

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Section: Q559k Channel Ion Selectivity Varies With Intracellular Ca 2mentioning

confidence: 99%

Section: Intracellular Ca 2+ and Membrane Depolarization Synergisticamentioning

confidence: 99%

See 2 more Smart Citations

Dynamic Change of Electrostatic Field in TMEM16F Permeation Pathway Shifts Its Ion Selectivity

Ye¹,

Han²,

He³

et al. 2019

Preprint

Self Cite

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“…Interestingly, Anoctamin family member ANO6 regulates the formation of a subset of extracellular vesicles (EVs) via phospholipid scrambling [22]. Furthermore, it has been shown that the chemical induction of giant plasma membrane vesicles (GPMVs) is dependent on ANO6 [23]. ANO7 has also been shown to act as a scramblase [15], and could thus play a similar role in vesicle formation as ANO6.…”

Section: Discussionmentioning

confidence: 99%

The interactome of the prostate-specific protein Anoctamin 7

Kaikkonen

Takala

Pursiheimo

et al. 2020

CBM

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BACKGROUND: Elevated Anoctamin 7 (ANO7) expression is associated with poor survival in prostate cancer patients. OBJECTIVE: The aim was to discover proteins that interact with ANO7 to understand its functions and regulatory mechanisms. METHODS: The proximity-dependent biotin identification (BioID) method was utilized. ANO7 fused to biotin ligase was transiently transfected into LNCaP cells, and the biotinylated proteins were collected and analysed by mass spectrometry. Four identified proteins were stained with dual fluorescent immunostaining and visualized using Stimulated emission depletion microscopy (STED). RESULTS: After bioinformatic filtering steps, 64 potentially ANO7-interacting proteins were identified and analysed with the GO enrichment analysis tool. One of the most prominently enriched cellular components was cellular vesicle. Co-localization was showed for staphylococcal nuclease and tudor domain containing 1 (SND1), heat shock protein family A (Hsp70) member 1A (HSPA1A), adaptor related protein complex 2 subunit beta 1 (AP2B1) and coatomer protein complex subunit gamma 2 (COPG2). CONCLUSIONS: This is the first study in which ANO7 interacting proteins have been identified. Although further studies are needed, the findings reported here expand our understanding of the role and regulation of ANO7 in prostate cancer cells. Furthermore, these results are likely to introduce new targets for the novel cancer therapies.

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Cell‐Derived Vesicles with Increased Stability and On‐Demand Functionality by Equipping Their Membrane with a Cross‐Linkable Copolymer

Huang

Hürlimann

Spanke

et al. 2022

Adv Healthcare Materials

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Cell‐derived vesicles retain the cytoplasm and much of the native cell membrane composition. Therefore, they are attractive for investigations of membrane biophysics, drug delivery systems, and complex molecular factories. However, their fragility and aggregation limit their applications. Here, the mechanical properties and stability of giant plasma membrane vesicles (GPMVs) are enhanced by decorating them with a specifically designed diblock copolymer, cholesteryl‐poly[2‐aminoethyl methacrylate‐b‐poly(ethylene glycol) methyl ether acrylate]. When cross‐linked, this polymer brush enhances the stability of the GPMVs. Furthermore, the pH‐responsiveness of the copolymer layer allows for a controlled cargo loading/release, which may enable various bioapplications. Importantly, the cross‐linked‐copolymer GPMVs are not cytotoxic and preserve in vitro membrane integrity and functionality. This effective strategy to equip the cell‐derived vesicles with stimuli‐responsive cross‐linkable copolymers is expected to open a new route to the stabilization of natural membrane systems and overcome barriers to biomedical applications.

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Chemically induced vesiculation as a platform for studying TMEM16F activity

Cited by 33 publications

References 63 publications

Dynamic Change of Electrostatic Field in TMEM16F Permeation Pathway Shifts Its Ion Selectivity

Dynamic Change of Electrostatic Field in TMEM16F Permeation Pathway Shifts Its Ion Selectivity

The interactome of the prostate-specific protein Anoctamin 7

Cell‐Derived Vesicles with Increased Stability and On‐Demand Functionality by Equipping Their Membrane with a Cross‐Linkable Copolymer

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