Acidic Amino Acids in the First Intracellular Loop Contribute to Voltage- and Calcium- Dependent Gating of Anoctamin1/TMEM16A

Xiao, Qinghuan; Cui, Yuanyuan

doi:10.1371/journal.pone.0099376

Cited by 20 publications

(28 citation statements)

References 42 publications

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“…The affinity of mouse Ano1(a,c) for Ca 2ϩ was 15-fold lower when segment c was deleted, which is a larger effect than the twofold change that we observed here in our studies on the full-length human Ano1(0,a,b,c,d) construct (40). Furthermore, compared with Ano1(a,c), Ano1(a)⌬EAVK had an increased SLOW without change to FAST , suggesting that EAVK sped up the slowgating mode of Ano1 activation (38). These studies suggest that EAVK contributes to but is not necessary for voltage and calcium sensitivity of Ano1, but the effects are variable depending on the specific Ano1 construct used and intracellular Ca 2ϩ concentration employed.…”

Section: Discussioncontrasting

confidence: 51%

EAVK segment “c” sequence confers Ca²⁺-dependent changes to the kinetics of full-length human Ano1

Strege

Gibbons

Mazzone

et al. 2017

American Journal of Physiology-Gastrointestinal and Liver Physi

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Anoctamin1 (Ano1 and TMEM16A) is a calcium-activated chloride channel specifically expressed in the interstitial cells of Cajal (ICC) of the gastrointestinal tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full-length human Ano1 sequence includes an additional exon, exon "0," at the NH terminus. Ano1 with exon 0 [Ano1] had a lower EC for intracellular calcium ([Ca]) and faster chloride current () kinetics. The Ano1 alternative splice variant with segment "c" encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alter at low [Ca] Exon 13 is expressed in 75-100% of Ano1 transcripts in most human tissues but only 25% in the human stomach. Our aim was to determine the effect of EAVK deletion on Ano1 parameters. By voltage-clamp electrophysiology, we examined in HEK293 cells transiently expressing Ano1 with or without the EAVK sequence [Ano1ΔEAVK]. The EC values of activating and deactivating for [Ca] were 438 ± 7 and 493 ± 9 nM for Ano1 but higher for Ano1ΔEAVK at 746 ± 47 and 761 ± 26 nM, respectively. Meanwhile, the EC values for the ratio of instantaneous to steady-state were not different between variants. Congruently, the time constant of activation was slower for Ano1ΔEAVK than Ano1 currents at intermediate [Ca] These results suggest that EAVK decreases the calcium sensitivity of Ano1 current activation and deactivation by slowing activation kinetics. Differential expression of EAVK in the human stomach may function as a switch to increase sensitivity to [Ca] via faster gating of Ano1. Calcium-activated chloride channel anoctamin1 (Ano1) is necessary for normal slow waves in the gastrointestinal interstitial cells of Cajal. Exon 0 encodes the NH terminus of full-length human Ano1 [Ano1], while exon 13 encodes residues EAVK on its first intracellular loop. Splice variants lack EAVK more often in the stomach than other tissues. Ano1 without EAVK [Ano1ΔEAVK] has reduced sensitivity for intracellular calcium, attributable to slower kinetics. Differential expression of EAVK may function as a calcium-sensitive switch in the human stomach.

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Section: Discussioncontrasting

confidence: 51%

EAVK segment “c” sequence confers Ca²⁺-dependent changes to the kinetics of full-length human Ano1

Strege

Gibbons

Mazzone

et al. 2017

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…Mean current from 0.5-1.0 s after the start of each test pulse was expressed as a fraction of cell capacitance [I/C (in pA/pF)], which was estimated by the amount of whole cell capacitance compensation dialed in during the seal test. As previously published by our group and others, we used voltage steps of 1 s in duration (1,7,10,15,20,36,42). Our reason for this was twofold.…”

Section: Cloning Of Ano1 With and Without Exonmentioning

confidence: 99%

A novel exon in the human Ca²⁺-activated Cl⁻ channel Ano1 imparts greater sensitivity to intracellular Ca²⁺

Strege

Bernard

Mazzone

et al. 2015

American Journal of Physiology-Gastrointestinal and Liver Physi

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Anoctamin 1 (Ano1; TMEM16A) is a Ca2+-activated Cl− channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca2+ regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-“exon 0” upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca2+ sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(−0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl− currents between isoforms with varying concentrations of intracellular Ca2+, extracellular anions, or Cl− channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(−0) in response to varying intracellular Ca2+. The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(−0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(−0). In conclusion, human Ano1 containing exon 0 imparts its Cl− current with greater sensitivity to intracellular Ca2+ and CACC inhibitors.

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“…As with I Cl generated by endogenous CaCC, I Cl generated by activation of TMEM16A cannot be described by a single exponential function (Scudieri et al . ; Xiao & Cui, ). In contrast, TMEM16B I Cl follows a mono‐exponential time course and appears to reach steady‐state at depolarized potentials (Adomaviciene et al .…”

Section: Introductionmentioning

confidence: 99%

Gating modes of calcium‐activated chloride channels TMEM16A and TMEM16B

Cruz-Rangel

Jesús-Pérez

Contreras-Vite

et al. 2015

The Journal of Physiology

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Key pointsr Calcium-activated chloride channels TMEM16A and TMEM16B support important physiological processes such as fast block of polyspermy, fluid secretion, control of blood pressure and sensory transduction.r Given the physiological importance of TMEM16 channels, it is important to study how incoming stimuli activate these channels. Here we study how channels open and close and how the process of gating is regulated.r We show that TMEM16A and TMEM16B display fast and slow gating. These gating modes are regulated by voltage and external chloride.r Dual gating explains the complex time course of the anion current. r Residues within the first intracellular loop of the channel influence the slow gating mode. r Dual gating is an intrinsic property observed in endogenous calcium-activated chloride channels and could be relevant to physiological processes that require sustained chloride ion movement.Abstract TMEM16A and TMEM16B are molecular components of the physiologically relevant calcium-activated chloride channels (CaCCs) present in many tissues. Their gating is dictated by membrane voltage (V m ), intracellular calcium concentrations ([Ca 2+ ] i ) and external permeant anions. As a consequence, the chloride current (I Cl ) kinetics is complex. For example, TMEM16A I Cl activates slowly with a non-mono-exponential time course while TMEM16B I Cl activates rapidly following a mono-exponential behaviour. To understand the underlying mechanism responsible for the complex activation kinetics, we recorded I Cl from HEK-293 cells transiently transfected with either TMEM16A or TMEM16B as well as from mouse parotid acinar cells. Two distinct V m -dependent gating modes were uncovered: a fast-mode on the millisecond time scale followed by a slow mode on the second time scale. Using long (20 s) depolarizing pulses both gating modes were activated, and a slowly rising I Cl was recorded in whole-cell and inside-out patches. The amplitude of I Cl at the end of the long pulse nearly doubled and was blocked by 100 μM tannic acid. The slow gating mode was strongly reduced by decreasing the [Cl − ] o from 140 to 30 mM and by altering the sequence of the first intracellular loop. Mutating 480 RSQ 482 to AVK in the first intracellular loop of TMEM16B nearly abolished slow gating, but, mutating 448 AVK 451 to RSQ in TMEM16A has little effect. Deleting 448 EAVK 451 residues in TMEM16A reduced slow gating. We conclude that TMEM16 CaCCs have intrinsic V m -and Cl − -sensitive dual gating that elicits complex I Cl kinetics.

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Acidic Amino Acids in the First Intracellular Loop Contribute to Voltage- and Calcium- Dependent Gating of Anoctamin1/TMEM16A

Cited by 20 publications

References 42 publications

EAVK segment “c” sequence confers Ca²⁺-dependent changes to the kinetics of full-length human Ano1

EAVK segment “c” sequence confers Ca²⁺-dependent changes to the kinetics of full-length human Ano1

A novel exon in the human Ca²⁺-activated Cl⁻ channel Ano1 imparts greater sensitivity to intracellular Ca²⁺

Gating modes of calcium‐activated chloride channels TMEM16A and TMEM16B

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Acidic Amino Acids in the First Intracellular Loop Contribute to Voltage- and Calcium- Dependent Gating of Anoctamin1/TMEM16A

Cited by 20 publications

References 42 publications

EAVK segment “c” sequence confers Ca2+-dependent changes to the kinetics of full-length human Ano1

EAVK segment “c” sequence confers Ca2+-dependent changes to the kinetics of full-length human Ano1

A novel exon in the human Ca2+-activated Cl− channel Ano1 imparts greater sensitivity to intracellular Ca2+

Gating modes of calcium‐activated chloride channels TMEM16A and TMEM16B

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Product

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EAVK segment “c” sequence confers Ca²⁺-dependent changes to the kinetics of full-length human Ano1

EAVK segment “c” sequence confers Ca²⁺-dependent changes to the kinetics of full-length human Ano1

A novel exon in the human Ca²⁺-activated Cl⁻ channel Ano1 imparts greater sensitivity to intracellular Ca²⁺