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. 2012 Mar 30;110(7):990-9.
doi: 10.1161/CIRCRESAHA.112.264440. Epub 2012 Mar 6.

Explaining calcium-dependent gating of anoctamin-1 chloride channels requires a revised topology

Kuai Yu  1 , Charity Duran, Zhiqiang Qu, Yuan-Yuan Cui, H Criss Hartzell
Affiliations

VSports最新版本 - Explaining calcium-dependent gating of anoctamin-1 chloride channels requires a revised topology

V体育ios版 - Kuai Yu et al. Circ Res. .

V体育ios版 - Abstract

Rationale: Ca2+ -activated Cl channels play pivotal roles in the cardiovascular system VSports手机版. They regulate vascular smooth muscle tone and participate in cardiac action potential repolarization in some species. Ca2+ -activated Cl channels were recently discovered to be encoded by members of the anoctamin (Ano, also called Tmem16) superfamily, but the mechanisms of Ano1 gating by Ca2+ remain enigmatic. .

Objective: The objective was to identify regions of Ano1 involved in channel gating by Ca2+ V体育安卓版. .

Methods and results: The Ca2+ sensitivity of Ano1 was estimated from rates of current activation, and deactivation in excised patches rapidly switched between zero and high Ca2+ on the cytoplasmic side. Mutation of glutamates E702 and E705 dramatically altered Ca2+ sensitivity. E702 and E705 are predicted to be in an extracellular loop, but antigenic epitopes introduced into this loop are not accessible to extracellular antibodies, suggesting this loop is intracellular. Cytoplasmically applied membrane-impermeant sulfhydryl reagents alter the Ca2+ sensitivity of Ano1 E702C and E705C as expected if E702 and E705 are intracellular. Substituted cysteine accessibility mutagenesis of the putative re-entrant loop suggests that E702 and E705 are located adjacent to the Cl conduction pathway V体育ios版. .

Conclusions: We propose an alternative model of Ano1 topology based on mutagenesis, epitope accessibility, and cysteine-scanning accessibility. These data contradict the popular re-entrant loop model by showing that the putative fourth extracellular loop (ECL 4) is intracellular and may contain a Ca2+ binding site VSports最新版本. These studies provide new perspectives on regulation of Ano1 by Ca2+. .

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Conflict of interest statement

Disclosures

The authors state that they have no conflict of interest.

Figures

Figure 1
Figure 1
Immunofluorescent staining of mAno1 containing tandem HA epitopes inserted at various locations. (A) HA tags were inserted into mAno1-EGFP at amino acids 570, 614, 672, 700 and 824. After transient expression, non-permeabilzed intact cells were stained with antibody for HA epitope. Green: Ano1-GFP, Red: Anti-HA, and merged image. Duplicate cover slips were permeabilized prior to incubation with HA antibody (red: permeabilized, anti-HA). For each construct, images were acquired at the same gain and settings, but settings may differ between constructs that were imaged on different days. Raw images from the Zeiss Zen acquisition software from permeabilized and non-permeabilized cells were assembled in Adobe Photoshop CS5 and brightness and contrast adjusted for all 4 panels equally. Ano1 currents for each construct were recorded with 20 μmol/L Ca. Average peak amplitude at +100mV and the number of recorded cells are listed. (B) Topological models of mAno1. The locations of HA tags are indicated with red numbers. Left: re-entrant loop model. Right: revised model. The topology of the sequence depicted in grey remains in question.
Figure 2
Figure 2
Mutation of two critical amino acids, E702 and E705, dramatically affects Ca2+-gated but not voltage-gated current of mAno1. (A, B) Representative whole-cell recordings of Ano1 current in transfected HEK293 cells at the indicated free [Ca2+]. Voltage protocol is shown above A. (A) WT-mAno1. (B) E702Q/E705Q mutant mANO1. (C, D) Steady-state current-voltage (I-V) relationships for (C) WT-mAno1and (D) E702Q/E705Q mANO1with different [Ca]i (N=5–9). (E) I–V relationships for E702D, E702K, E705D and E705K mutants with 20 μmol/L Ca2+ (N=5–9).
Figure 3
Figure 3
Activation and deactivation kinetics of Ano1 with rapid Ca2+ perfusion in inside-out excised patches. (A–C) Representative traces of Ano1 current in response to application (left) and washout (right) of Ca2+ at the indicated holding potentials. (A) WT-mAno1. (B) E702Q mANO1. (C) E705Q mANO1. (D-F) Vm dependence of τon, τoff, and EC50 for WT Ano1 (open circles), E702Q (filled circles), and E705Q (filled triangles).
Figure 4
Figure 4
Effects of sulfhydryl modification on activation and deactivation kinetics of Ano1 in excised inside-out patches with rapid Ca2+ perfusion. Excised patches were switched between zero and high Ca2+. They were then exposed to MTS reagent in the presence of zero Ca2+ for 10 sec, MTS reagent was washed away, and the patch was switched from zero Ca2+ to high Ca2+ again. (A, B) Normalized current traces of E702C mAno1showing examples of changes in τon and τoff caused by MTSET+ (A) and MTSES− (B). Traces were normalized to the same maximal amplitude. As shown in Fig. S3, MTSET+ causes a decrease in current and MTSES− increases the current. The magnitude of the effect of MTS reagent on current amplitude depends where on the Ca2+dose-response curve the experiment is performed. (C–E) Lack of effect of MTS reagents on τon, τoff, and EC50 for WT Ano1. (F–H) Effects of MTS reagents on τon, τoff, and EC50 for E702C mAno1. Open circle: before application of MTS reagent. Filled circle: after MTSET+, triangle: after MTSES−. (N=3–6).
Figure 5
Figure 5
Effect of R621E mutation on anion:cation permeability of mAno1. (A, B) Whole-cell recordings of (A) WT mAno1 in symmetrical 150 mmol/L NaCl with 180 nmol/L Cai and (B) R621E mAno1 in symmetrical 150 mmol/L NaCl with 1.1 μmol/L Cai. The R621E mutation decreased the Ca2+ sensitivity of the channel, requiring a larger Ca2+ concentration to generate a measurable current. (C, D) Current-voltage relationships of (C) WT mAno1 and (D) R621E mAno1 with different extracellular [NaCl]. (E) Change in reversal potential (ΔErev) for different extracellular [NaCl] or [CsCl] determined from experiments like those in C and D. Lines are best fits to the Goldman-Hodgkin-Katz equation.
Figure 6
Figure 6
Essential cysteines in mAno1 and scanning cysteine accessibility of the proposed new TMD6 (amino acids 620–646). (A) Six extracellular cysteines are required for mAno1 function. Amplitudes of whole-cell currents activated by 20μmol/L [Ca2+]i at +100mV from WT and cysteine-substituted mutants of mAno1 expressed in HEK293 cells. Inset shows location of cysteines (solid red circles: essential; open red circles: not-essential for channel function) in the old model of mAno1. 6C: mAno1 with all cysteines except C370, C379, C383, C386, C395 and C836 replaced with serines. C836S is wild type mAno1 with C836 only replaced with serine. The other constructs have each of the essential cysteines in mAno16C as indicated replaced with serine. (B) Example of change in current amplitude of G629C mANO16C. MTSET-induced increase in current is not reversed by washout of MTSET but is reversed by 5 mmol/L DTT. (C) Average Ano1 currents recorded from WT mAno1, mAno16C and cysteine-substituted mAno16C. (D) Effects of MTSET+ (left), MTSES− (middle) and MTSEA+ (right) on cysteine-substituted mAno16C. The percent change of current at +100mV within 2 min of exposure to MTS reagent was calculated by [100 (Iafter MTS − Ibefore MTS)/Ibefore MTS]. Cysteine-substituted amino acids labeled with an asterisk were significantly (p < 0.05) affected by MTS reagents (N= 3–6).
Figure 7
Figure 7
Effects of replacement of amino acids in mANO16C with cysteine on I− permeability and conductance relative to Cl−. (A) Relative I− permeability was calculated from the shift in reversal potential after replacing extracellular Cl− with I−. (B) Relative I− conductance was measured as the ratio of slopes of the current-voltage relationship at the reversal potential after and before extracellular anion change (N= 3–10).
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