Activation of the Cl- channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor

Abstract

We report that anoctamin 1 (ANO1; also known as TMEM16A) Ca(2+)-activated Cl(-) channels in small neurons from dorsal root ganglia are preferentially activated by particular pools of intracellular Ca(2+). These ANO1 channels can be selectively activated by the G protein-coupled receptor (GPCR)-induced release of Ca(2+) from intracellular stores but not by Ca(2+) influx through voltage-gated Ca(2+) channels. This ability to discriminate between Ca(2+) pools was achieved by the tethering of ANO1-containing plasma membrane domains, which also contained GPCRs such as bradykinin receptor 2 and protease-activated receptor 2, to juxtamembrane regions of the endoplasmic reticulum. Interaction of the carboxyl terminus and the first intracellular loop of ANO1 with IP3R1 (inositol 1,4,5-trisphosphate receptor 1) contributed to the tethering. Disruption of membrane microdomains blocked the ANO1 and IP3R1 interaction and resulted in the loss of coupling between GPCR signaling and ANO1. The junctional signaling complex enabled ANO1-mediated excitation in response to specific Ca(2+)signals rather than to global changes in intracellular Ca(2+).

Fig. 1. Ca²⁺-activated Cl⁻ channels in small DRG neurons are preferentially activated by Ca²⁺ release from the ER

(A) Whole cell patch-clamp recording of currents in response to a voltage pulse from −80 to 0 mV in small DRG neurons. The recordings were made using TEACl-based extracellular and CsCl-based intracellular solutions. The green trace represents inhibition of VGCC by Cd²⁺ (100 μM) in the same cell in which the control (black) trace was recorded. The box inset depicts the response of the same cell to capsaicin (CAP; 1 μM) at a holding potential of −60 mV. 19/20 and 1/20 represent the number of neurons exhibiting the specified type of response. (B) BK (1 μM)-induced inward current in a small DRG neuron when recorded in extracellular solution with and without Ca²⁺. (C) PAR2-PL (10 μM)-induced inward current in a small DRG neuron; in control conditions or following pretreatment with thapsigargin (Tg, 2 μM, 3 min); NFA, niflumic acid (100 μM). (D, E) Summary data for (B) and (C), respectively. Number of responsive neurons out of total neurons tested is indicated within each bar.

Fig. 2. Imaging CaCC activation with a halide-sensitive EYFP

(A) Fluorescence intensity of a DRG neuron transfected with H148Q/I152L EYFP was monitored during application of bath solution containing 30 mM NaI on its own or in combination with 1 μM BK (as indicated by the black bars). Pseudocolored images (lower panel) were taken at times indicated by the Roman numerals. (B) Averaged time courses of normalized fluorescence (F/F₀) of H148Q/I152L EYFP-transfected neurons perfused with 30 mM NaI-containing extracellular solution either alone (n = 10) or in combination with 1 μM BK (n = 15); with 1 μM BK and 100 μM NFA (n = 8); or with 50 mM KCl (n = 27). The time of compound application is indicated by the shaded area. ** p≤0.01 as compared to vehicle alone.

Fig. 3. ANO1 channels form signaling complexes with IP₃ receptors and GPCRs

(A) Top: Traces of inward currents measured in neurons in response to PAR-2-PL (10 μM) subjected to whole-cell dialysis with BAPTA (10 mM, 4 min) or EGTA (10 mM, 4 min) or control (no calcium chelator). Holding potential was −60 mV. Bottom: Summary and quantification of the results. (B, C) Immunoprecipitation of ANO1 by an antibody against IP₃R1 (B) and of IP₃R1 by an antibody against ANO1 (C) from lysates of whole DRG from rat. Control immunoprecipitations were performed using mouse or goat IgG as appropriate. IP: and WB: indicate the antibodies used for immunoprecipitation and Western blotting, respectively; co-IP, coimmunoprecipitation. (D) Immunoprecipitation of B₂R (upper panel) and PAR-2 (middle panel) by an antibody against IP₃R1. Control immunoprecipitations were performed using mouse IgG. E, immunoprecipitation of B₂R (upper panel) and PAR-2 (middle panel) receptors by an antibody against Cav-1. Control immunoprecipitations were performed using mouse IgG. In B-E, all results shown are representative of three independent experiments and 5% of total lysate protein was used for input (Lysate).

Fig. 4. PLA indicates that ANO1 and IP₃R1 are in close proximity

(A) Schematic illustrating principle of PLA. When two proteins are in close proximity, the antibodies connected to specific DNA fragments are close enough to complementary bind to a connector oligonucleotide, which then forms a circular structure amplified in a rolling cycle amplification (RCA) process. The RCA product is detected by hybridization of dye-conjugated oligonucleotides complementary to a tag sequence in the RCA product (43). (B) Punctate ANO1-IP₃R1 PLA signal in small-diameter DRG neuron but not in surrounding glia. Image on the left is a bright-field illumination, middle image shows DAPI staining; image on the right shows the PLA signal as detected with a 488 nM argon laser; representative of three independent preparation. (C) Absence of ANO1-IP₃R1 PLA signal in HUVECs (left image shows DAPI staining, right image shows PLA staining); representative of two independent preparations. (D) Absence of ANO1-VGCC PLA signal in DRG cultures (neurons and glia); pan-VGCC antibody was used to immunolabel VGCC; representative of two independent preparations.

Fig. 5. ANO1 localizes to lipid rafts in DRG neurons

(A) Sucrose density gradient fractionation of detergent extracts of rat DRG immunoblotted for Cav-1 (lipid raft marker), ANO1, B₂R, PAR-2 and transferrin receptor (CD71, non-raft-localized protein) under control conditions or following cholesterol extraction of the lysates with 50 mM methyl-β-cyclodextrin (βMCD). Each experiment was repeated at least three times. (B) Immunoprecipitation of ANO1 (upper blot) or B₂R (lower blot) with IP₃R1 from control DRG lysate or in DRG lysate treated with βMCD. Bar chart summarizes densitometry data from three independent experiments. Mean optical densities from identical areas around each coimmunoprecipitation band (upper blots) were normalized to the density of the corresponding WB band (lower blots); the density of the bands in βMCD-treated samples is expressed as a fraction of control. Results are representative of three or more independent experiments; * p≤0.05. (C) Effect of βMCD treatment on ANO1-IP₃R1 interaction detected by PLA in DRG neurons. DAPI and PLA images are shown for control and βMCD-treated cultures as indicated. The bar chart summarizes the number of puncta per PLA-positive neuron (control, n = 14; βMCD, n = 14). *** p≤0.001

Fig. 6. ANO1 and IP₃R1 are engaged in functional interaction

(A) GST pull-down experiments. Top panel is a schematic depiction of ANO1 channel. Below left is a schematic depiction of GST-fusion proteins containing the C-terminus (residues 963-1040, ‘C’), the loop between the second and third transmembrane domains (residues 505-568, ‘L’) and the N-terminus (residues 1-407, ‘N’) of ANO1. Below right: upper Western blot shows the purified GST-fusion peptides (detected with the antibody against GST); lower panel shows a pull-down experiment with the indicated peptides and IP₃R1 from the DRG lysate (detected with the antibody against IP₃R1). All results shown are representative of three independent experiments. (B) The effect of the three cytosolic domains of ANO1 on PAR2-PL-induced inward currents in DRG neurons. Each ANO1 cytosolic domain was individually overexpressed in DRG neurons and inward current was tested by patch clamp. Traces are representative recordings from cells transfected with the indicated constructs. Vector is EGFP only. Periods of PAR2-PL (10 μM) application are indicated by black bar. (C, D) Bar charts summarizing the current amplitudes (C) and proportions of the neurons displaying inward current (D) in the indicated neuron groups.

Fig. 7. Disruption of ANO1-containing complexes results in coupling of CaCC activity to ‘global’ Ca²⁺ elevations and overexcitable neurons

(A) Whole cell patch-clamp experiments showing effect of treatment of cultured DRG neurons with 10 mM βMCD for 30 min on inward tail current. Voltage protocol and labelling as in Fig. 1A. (B) Summary for control, βMCD, and αCD (10 mM, 30 min) experiments like those shown in (A) for βMCD-treated neurons. I_CaCC-VGCC was calculated as a difference in peak tail current amplitudes after the depolarizing pulses with and without Ca²⁺ influx; neurons were considered as not displaying activation of CaCC by VGCC when the resulting amplitude was below 40 pA. Red horizontal bars represent mean values of all neurons tested in each group. Numerals above and below the dotted line represent number of neurons with and without VGCC-induced CaCC, respectively. (C) Effect of βMCD treatment on I⁻ influx induced by depolarization with 50 mM KCl in DRG neurons. Averaged time courses of normalized fluorescence (F/F₀) of H148Q/I152L EYFP-transfected neurons perfused with 30 mM NaI-containing extracellular solution either alone (vehicle, n = 7) or in neurons treated with βMCD (10 mM, 30 min) and then stimulated with 50 mM KCl (High K⁺, n = 10) or 50 mM KCl and NFA (100 μm) (High K⁺ +NFA, n=5). The time of application of NaI and the depolarizing stimulus is indicated by the shaded area. Dotted grey line represent mean data for the effect of 50 mM KCl in control (βMCD untreated) neurons; taken from the Fig. 2B for comparison. (D) Current clamp experiments showing action potentials of control DRG neurons or neurons treated with βMCD in response to injection of a 600 pA depolarizing current pulse (depicted under the traces). (E) Summary for the experiments like those shown in (D); labelling as in (B) for neurons exposed to βMCD in the presence of high or low intracellular Cl⁻ or in neurons exposed to αCD. (F) Summary of the effects of βMCD or αCD treatment on the GPCR-induced CaCC. (G) Exemplary current traces from the experiments summarized in (F). (H) Simplified scheme of the proposed juxtamembrane arrangements within an ANO1-containing signaling microdomain. Gray ovals represent Cav-1.