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Molecular underpinning of intracellular pH regulation on TMEM16F
Pengfei Liang1, Huanghe Yang1,2
1Department of Biochemistry, Duke University Medical Center, Durham, NC.
Abstract:
TMEM16F, a dual-function phospholipid scramblase and ion channel, is important in blood coagulation, skeleton development, HIV infection, and cell fusion. Despite advances in understanding its structure and activation mechanism, how TMEM16F is regulated by intracellular factors remains largely elusive. Here we report that TMEM16F lipid scrambling and ion channel activities are strongly influenced by intracellular pH (pHi). We found that low pHi attenuates, whereas high pHi potentiates, TMEM16F channel and scramblase activation under physiological concentrations of intracellular Ca2+ ([Ca2+]i). We further demonstrate that TMEM16F pHi sensitivity depends on [Ca2+]i and exhibits a bell-shaped relationship with [Ca2+]i: TMEM16F channel activation becomes increasingly pHi sensitive from resting [Ca2+]i to micromolar [Ca2+]i, but when [Ca2+]i increases beyond 15 µM, pHi sensitivity gradually diminishes. The mutation of a Ca2+-binding residue that markedly reduces TMEM16F Ca2+ sensitivity (E667Q) maintains the bell-shaped relationship between pHi sensitivity and Ca2+ but causes a dramatic shift of the peak [Ca2+]i from 15 µM to 3 mM. Our biophysical characterizations thus pinpoint that the pHi regulatory effects on TMEM16F stem from the competition between Ca2+ and protons for the primary Ca2+-binding residues in the pore. Within the physiological [Ca2+]i range, the protonation state of the primary Ca2+-binding sites influences Ca2+ binding and regulates TMEM16F activation. Our findings thus uncover a regulatory mechanism of TMEM16F by pHi and shine light on our understanding of the pathophysiological roles of TMEM16F in diseases with dysregulated pHi, including cancer.
Insights
Intracellular pH (pHi) regulates TMEM16F channel and scramblase activity, with low pHi attenuating and high pHi potentiating function. This pH sensitivity is dependent on intracellular calcium concentrations, revealing a new regulatory mechanism for TMEM16F.
Area of Science:
- Biophysics
- Cell Biology
- Molecular Biology
Background:
- TMEM16F is a dual-function protein with phospholipid scramblase and ion channel activities.
- It plays crucial roles in various physiological processes, including blood coagulation, skeleton development, HIV infection, and cell fusion.
- The regulation of TMEM16F by intracellular factors, particularly pH, is not well understood.
Purpose of the Study:
- To investigate the influence of intracellular pH (pHi) on TMEM16F lipid scrambling and ion channel activities.
- To elucidate the relationship between pHi sensitivity, intracellular calcium concentrations ([Ca2+]i), and TMEM16F function.
- To identify the molecular mechanisms underlying pHi regulation of TMEM16F.
Main Methods:
- Patch-clamp electrophysiology to measure TMEM16F ion channel activity.
- Lipid scrambling assays to assess TMEM16F scramblase function.
- Site-directed mutagenesis to investigate the role of specific residues in Ca2+ binding and pHi sensitivity.
- Biophysical characterization of TMEM16F activity across a range of pHi and [Ca2+]i conditions.
Main Results:
- TMEM16F channel and scramblase activities are modulated by pHi, with low pHi attenuating and high pHi potentiating function under physiological [Ca2+]i.
- TMEM16F pHi sensitivity exhibits a bell-shaped dependence on [Ca2+]i, peaking at micromolar concentrations and diminishing at higher [Ca2+]i.
- A Ca2+-binding mutant (E667Q) retained the bell-shaped pHi sensitivity but shifted the peak [Ca2+]i to millimolar levels.
- Proton competition with Ca2+ for binding sites in the TMEM16F pore underlies the observed pHi regulation.
Conclusions:
- Intracellular pH is a significant regulator of TMEM16F activity, influencing both its ion channel and scramblase functions.
- The interplay between pHi and [Ca2+]i is critical for TMEM16F activation.
- These findings provide insights into the pathophysiological roles of TMEM16F in diseases associated with altered pHi, such as cancer.
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