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, 467 (8), 1677-87

Two Helices in the Third Intracellular Loop Determine Anoctamin 1 (TMEM16A) Activation by Calcium

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Two Helices in the Third Intracellular Loop Determine Anoctamin 1 (TMEM16A) Activation by Calcium

Jesun Lee et al. Pflugers Arch.

Abstract

Anoctamin 1 (ANO1)/TMEM16A is a Cl(-) channel activated by intracellular Ca(2+) mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca(2+). Here, we demonstrate that two helices, "reference" and "Ca(2+) sensor" helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca(2+)-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca(2+)-dependent activation because neutralization of these charges change the Ca(2+) sensitivity. We now predict that the Ca(2+) sensor helix attaches to the reference helix in the resting state, and as intracellular Ca(2+) rises, Ca(2+) acts on the sensor helix, which repels it from the reference helix. This Ca(2+)-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.

Figures

Fig. 1
Fig. 1
ANO1 activation by Ca2+ requires the third intracellular loop (ICL3). a Proposed topologies of ANO1, conventional and revised forms. ED, Glu, and Asp-rich region; EF EF-hand region; ICL1 and ICL3 the first and the third intracellular loops, respectively. b Example traces of channel currents of inside-out patches isolated from ANO1, ANO2, ANO1 deletion mutants, and EGFP-transfected HEK 293T cells. Δ5E a ANO1 deletion mutant at 444-EEEEE-448, ΔICL3 a ANO1 deletion mutant of the third intracellular loop region. Holding potential (E h) = +80 mV. c Concentration-response relationships of channel currents of ANO1, ANO2, and ANO1 mutants activated by various concentrations of Ca2+. Each current is normalized to the maximal response. Lines are fitted to the Hill equation, I/Imax = 1/(1 + [Ca2+]/EC50)n. EC50s (in μM) for ANO1 (red circle), ANO2 (pink diamond), and ANO1 mutants were 0.7 (n = 11), 9.8 (n = 13), 120-HQNNKRFRRQQYQGNLLEAGLQLQNDQDT-148 (ED9 (green triangle), 2.4, n = 9), Δ5E (blue circle, 2.1, n = 8), 285-AGAYAGA-291 (brown inverted triangle, 17.8, n = 6), and ΔICL3 (light blue square, 2,509, n = 10), respectively. (Right) The I/V curves of WT ANO1 at various concentration of Ca2+
Fig. 2
Fig. 2
Homology models of the ICL3 regions of ANO1 and ANO2. a A crystal structure of a part of a peptidoglycan deacetylase of Helicobacter pylori that shares structural homology with the ICL3 region of ANO2. b A homology model of the ICL3 of ANO2. Structure of the ICL3 region of ANO2 was modeled using the backbone coordinates of a deacetylase of Helicobacter pylori as a template. Side chains of Arg and Lys are shown in stick. Blue and red atoms in the side chains represent nitrogen and oxygen atoms, respectively. c A homology model of the ICL3 of ANO1. Structure of the ICL3 region of ANO1 was modeled using the backbone coordinates of the ICL3 region of ANO2 as a template, (right) top view. (d) Sequence alignment of the ICL3 regions of ANO1 and ANO2
Fig. 3
Fig. 3
Circular dichroism spectra of two reference helix peptides of ANO1 and ANO2. The two peptides that span the reference helices in the ICL3 regions of ANO1 and ANO2 were 652-LFEIGIPKMKKFIRYLKL-669 and 604-IFEIGVPKLKKLFRKLKD-621, respectively. Note a typical α-helical character with a positive band around 192 nm and negative bands around 208 and 222 nm
Fig. 4
Fig. 4
The reference helix of the ICL3 region interacts with the Ca2+ sensor helix in a Ca2+-dependent way. a, b Physical interaction between the reference and Ca2+ sensor helices was assessed with a surface plasmon resonance assay. Biotinylated Ca2+ sensor helix peptide (692-NLEPFAGLTPEYMEM-706) was immobilized on a streptavidin-coated gold sensor chip. Resonance intensities were measured after solutions containing 0, 0.4, and 2.0 mM Ca2+ and various concentrations of reference helix peptide (651-NLFEIGIPKMKKFIRYLKLRR-671) (a) or its Ala-substituted mutants (651-NLFEIGIPAMAAFIAYLALAA-671) (b) were flowed through the gold chip. Note that the wild-type reference helix peptide shows a binding with the Ca2+ sensor helix in a Ca2+-dependent manner (a), which is not observed for the Ala-substituted mutant peptide (b). c Ca2+-induced ANO1 currents were blocked by bath application of the reference helix peptide (651-NLFEIGIPKMKKFIRYLKLRR-671, 4 μM) to inside-out patches but not by the Ala-mutant peptide (651-NLFEIGIPAMAAFIAYLALAA-671, 4 μM) (lower panel). Ca2+ (3 μM) was applied three times to inside-out patches of ANO1-transfected HEK cells. At the second Ca2+ challenge, the peptides were also given. E h = +80 mV. d A summary plot of the effects of applications of reference helix and Ca2+ sensor helix peptides on Ca2+-activated ANO1 currents. Current amplitudes were normalized to the current amplitude obtained after of the first Ca2+ challenge. At the second Ca2+ challenge, reference helix peptide (black circle, **p < 0.01 compared to the relative response of vehicle application, one-way ANOVA, Newman-Keuls post hoc test, n = 13), Ala-replaced reference helix peptide (black inverted triangle, n = 8), the Ca2+ sensor helix peptide (700-TPEYMEMIIQFGF-712) (black square, **p < 0.01 compared to the relative response of vehicle application, one-way ANOVA, Newman-Keuls post hoc test, n = 7), and vehicle (white circle, n = 8) were also applied
Fig. 5
Fig. 5
Mutations in the ICL3 regions of ANO1 and ANO2 shift the sensitivity of Ca2+-dependent activation. a Replacement of Arg, Lys, or Glu residue with Ala, Gly, or Gln residue in the reference and Ca2+ sensor helices caused rightward shifts in the dose-response relationship curve of ANO1. EC50s (in μM) for ANO1 mutants were 659-QMKK-662 (pink square, 6.1, n = 4), KMQK (blue circle, 4.7, n = 5), KMKQ (green triangle, 3.6, n = 6), GMGG (dark green square, 22.8, n = 6), 665-GYLG-668 (light blue square, 6.2, n = 6), deletion of 659-KMKKFIRYLK-668 (gray inverted triangle, 51.0, n = 9), 670-GGQ-672 (brown inverted triangle, 1.2, n = 7), and 702-QYMQ-705 (blue square 11260, n = 7). The dose-response curve of wild-type (WT) ANO1 is the same as shown in Fig. 1c (red line). b Mutations in the ICL3 region of ANO2 also shifted dose-response curves rightward. EC50s were 611-QLQQ-614 (red square, 50.2 μM, n = 7), 611-GLGGLFGGLG-620 (green triangle, 348.9 μM, n = 8), and 654-AYMA-657 (light blue circle, 3.3 mM, n = 6). The dose-response curve of WT ANO2 (blue line) is same as shown in Fig. 1c
Fig. 6
Fig. 6
Eact, a synthetic agonist of ANO1 acts on the ICL3 region. a Example traces depicting activation by Eact of WT ANO1 (upper left), 444-EEEEE-448 (Δ5E, upper right), ΔICL3 (lower left), and 659-QMQQFIAYLQ-668 mutant (lower right). b Example traces of Eact-induced ANO1 currents, which were blocked by bath application of the reference helix peptide (Ref helix peptide, 651-NLFEIGIPKMKKFIRYLKLRR-671, 4 μM, lower panel) but not by the Ca2+ sensor helix peptide (CSH peptide, 700-TPEYMEMIIQFGF-712, 10 μM, middle panel). To test the effects of peptide, 1 μM Eact was applied three times. At the second Eact challenge, each peptide was also applied. c A summary of the effects of applications of helix peptides on Eact-induced ANO1 currents. Current amplitudes were normalized to the current amplitude obtained after of the first Eact challenge. Reference helix peptide (black square, n = 5), its Ala-mutant peptide (black inverted triangle, n = 5), Ca2+ sensor helix peptide (black triangle, n = 5), or vehicle (white circle, n = 14) was applied at the second Eact challenge. *p < 0.05 compared to the relative response of vehicle application, one-way ANOVA followed by Newman-Keuls post hoc test
Fig. 7
Fig. 7
The two helices in the ICL3 region are dispensable for voltage- and heat-induced ANO1 activation. a Current responses of WT ANO1 to voltage pulses (−200 to +200 mV in 20-mV increment) at 0.1, 1.0, and 10 μM Ca2+ (left). A G–V curve of WT ANO1 at 0.3, 1.0, 3.0, and 10 μM Ca2+ (right). G/Gmaxs at different [Ca2+]i were plotted against membrane potential (V m). b, c G–V curves of various mutants in the reference helix at 10 μM [Ca2+]i (b) and an ICL3-deleted mutant (residues 653–711) of ANO1 at 10 μM ~ 10 mM [Ca2+]i (c). The G–V curve of WT ANO1 was the same as shown in Fig. 7a (black line). d Traces of heat-induced whole-cell currents of EGFP-, WT-, ΔICL3-, and Δreference helix mutant (Δ659-668)-transfected HEK 293T cells. E h = −60 mV. e A summary of heat-induced currents of ANO1 and its mutants. f A schematic diagram depicting a molecular mechanism underlying Ca2+-dependent activation of ANO1 at the ICL3 region

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