Journal: Nature Communications

Article Title: Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca 2+

doi: 10.1038/s41467-025-58682-w

Figure Lengend Snippet: A – D Parotid acini from 3 wild-type and 3 IRBIT −/− mice were used to measure Ca 2+ in response to 10 nM ( A , B ) or 100 µM carbachol ( C , D ) in isolated acinar clusters. Ca 2+ oscillations (example traces in A ) were analyzed in 4 experiments and 15 wild-type and 4 experiments, and 19 IRBIT −/− acinar clusters to obtain the number of cells showing Ca 2+ oscillations and total amount of Ca 2+ increase ( B ). For Ca 2+ release and influx ( C , D ), the acini were stimulated first in Ca 2+ -free media, and then Ca 2+ was added back. Average traces with acini from one mouse are shown in ( C ), and the Ca 2+ release and influx in 30 acinar clusters from 3 mice of each line are shown in ( D ). E Saliva secretion was stimulated by 0.125 mg/kg pilocarpine in 4 wild-type and 3 IRBIT −/− mice. F , G Example traces of intracellular Cl - measurements with the Cl - dye MQAE and determined as F/F 0 in wild-type acini treated with vehicle (black trace) or 5 µM Ani9 (red trace) and stimulated with 100 µM carbachol ( F ) or acini from wild-type (black/green traces) or IRBIT −/− acini (blue, red traces) stimulated with 100 µM carbachol ( G ). ( H – J ), Dose-response for carbachol-stimulated ANO1 activity in acini from wild-type (black) or IRBIT −/− mice (red) ( H ). Results from 6 independent experiments were fitted to a Michaelis-Menten equation to obtain the Vmax ( I ) and Apparent Km for carbachol ( J ). All results are shown as mean ± s.e.m.

Article Snippet: The perfusate was switched to a Cl - -free solution by replacing Cl - in bath solution with NO 3 - , and after determining the baseline of MQAE fluorescence, the acini were stimulated with carbachol in the presence and absence of the ANO1 inhibitor Ani9 (5 μM, Cayman Chemical).

Techniques: Isolation, Activity Assay

Journal: Nature Communications

Article Title: Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca 2+

doi: 10.1038/s41467-025-58682-w

Figure Lengend Snippet: A – C Shown are the Ca 2+ dependence of ANO1 (black, blue), in the presence of IRBIT (green, red) and F/I stimulation (blue, red) ( A ), the Vmax ( B ) and apparent Km for Ca 2+ ( C ) from 4 independent experiments. D Surface expression of ANO1 in cells transfected with vector or IRBIT (red) was assayed by biotinylation, with example blots and the average of 4 independent experiments. E Analysis of surface ANO1 by TIRF in 6 independent experiments in the presence and absence of IRBIT and F/I. F Current was measured in 4 independent experiments with a pipette solution containing 1 mM ATP and 0.3 µM Ca 2+ . G Current was measured in 5 independent experiments in cells expressing ANO1 (black) and IRBIT (green) and stimulated with F/I (red); ANO1(S673A), IRBIT and stimulated with F/I (dotted columns); ANO1(S673D), IRBIT and stimulated with F/I (filled columns). H89 0.1 µM (brown) was included in the pipette solution. H ANO1 levels were measured by TIRF in 4 independent experiments in cells expressing ANO1, ANO1(S673A), or ANO1(S673D) as indicated, and empty vector (black), IRBIT (green) and stimulated with F/I (Blue, red). I – K Shown are the Ca 2+ dependence of ANO1 and ANO1(S673D) stimulated with F/I (blue, green) ( I ), the Vmax ( I ) and Km for Ca 2+ ( J ) from 4 independent experiments. L FRB+5PIPase-FKBP and rapamycin were used to deplete PI(4,5)P 2 and measure ANO1 current in the presence of IRBIT and F/I. M A Schematic illustrating the ANO1-associated tether and cAMP/PKA pathway proteins that form regulatory complexes at the ER/PM junctions, as examined in the present studies, is shown. In ( G ) pipette solution Ca 2+ was 0.3 µM. In these and other experiments below, unless otherwise indicated, experiments were with pipette solution in which Ca 2+ was buffered to 0.3 µM. The 0.3 µM Ca 2+ was selected since the Ca 2+ -dependent current can be clearly resolved, and the effect of the various regulators was optimal. All results are shown as mean ± s.e.m.

Article Snippet: The perfusate was switched to a Cl - -free solution by replacing Cl - in bath solution with NO 3 - , and after determining the baseline of MQAE fluorescence, the acini were stimulated with carbachol in the presence and absence of the ANO1 inhibitor Ani9 (5 μM, Cayman Chemical).

Techniques: Expressing, Transfection, Plasmid Preparation, Transferring

Journal: Nature Communications

Article Title: Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca 2+

doi: 10.1038/s41467-025-58682-w

Figure Lengend Snippet: A ANO1 current was measured in 4 independent experiments in the presence of VAPA (blue) and F/I, IRBIT, IRBIT + VAPA and stimulated with F/I as indicated in the columns; Cells treated with siVAPA (filled columns), IRBIT, and stimulated with F/I; Celle expressing VAPA + E-Syt1 and stimulated with F/I. B Current was measured in cells transfected with ANO1 (black) and either IRBIT (open columns) or IRBIT(Y75A/A78E/S84E) FFAT site mutant (filled columns) and VAPA (blue and light blue) and stimulated with F/I. The results are from 4 independent experiments. C ANO1 was expressed in IRBIT −/− cells alone (black) and with VAPA (green) and stimulated with F/I (blue, red). The results are from 3 independent experiments. D – F Effect of VAPA (black) and VAPA + IRBIT (blue) before and after treatment with F/I (red, green) on ANO1 surface expression that was measured by biotinylation in 3 independent experiments. Example blots are in ( D ) and averages relative to total ANO1 and to GAPDH are in ( E ) and ( F ), respectively. G – I The Ca 2+ dependence of the ANO1(black) and ANO1(S243E) (red) stimulated with F/I (green) ( G ), the Vmax ( H ) and apparent Km for Ca 2+ ( I ) were obtained in 3 independent experiments. J – L The Ca 2+ dependence of the ANO1 in the presence of vector (black), VAPA (red) and stimulated with F/I (blue), VAPA + IRBIT (dark yellow), and stimulated with F/I (green) ( J ), the Vmax ( K ) and apparent Km for Ca 2+ ( L ) were obtained from 4 independent experiments. M Current was measured in pipette solution with Ca 2+ buffered to 0.3 (5 independent experiments) or 10 µM (4 independent experiments) in cells transfected with ANO1 and VAPA (black, red, orange) or VAPA + IRBIT (blue, green, dark yellow) and stimulated with F/I (filled columns). N – P The Ca 2+ dependence of the ANO1+VAPA (red, blue) treated with siAC6 (black, grean) and stimulated with F/I (blue, green) ( N ), the Vmax ( O ), and apparent Km for Ca 2+ ( P ). In ( A – C , M ) pipette solution, Ca 2+ was 0.3 µM. Results are from 6 (VAPA + siAC6) or 3 (all others) independent experiments. All results are shown as mean ± s.e.m.

Article Snippet: The perfusate was switched to a Cl - -free solution by replacing Cl - in bath solution with NO 3 - , and after determining the baseline of MQAE fluorescence, the acini were stimulated with carbachol in the presence and absence of the ANO1 inhibitor Ani9 (5 μM, Cayman Chemical).

Techniques: Expressing, Transfection, Mutagenesis, Plasmid Preparation, Transferring

Journal: Nature Communications

Article Title: Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca 2+

doi: 10.1038/s41467-025-58682-w

Figure Lengend Snippet: A Current was measured in cells expressing ANO1 (black) with E-Syt1 (red), E-Syt2 (green), or E-Syt3 (blue) and stimulated with F/I (filled columns). The results are from 4 independent experiments. B Current was measured in cells treated with scrambled siRNA (open columns), siE-Syt1 (filled columns), or siE-Syt2 strips columns) and transfected with ANO1 (black) and IRBIT (red) and stimulated with F/I (green). The results are from 4 independent experiments. C Current was measured in cells expressing ANO1 (open columns) or ANO1 + IRBIT (close columns) and E-Syt1 (green) and treated with F/I (red). The results are from 3 independent experiments. D Effect of E-Syt1 and F/I on E-Syt1-ANO1 Co-IP and on surface ANO1 expression. This is one of 3 similar experiments. E Rescue by IRBIT of ANO1 current measured in IRBIT −/− cells, as indicated. The results are from 5 independent experiments. F Current was measured in cells treated with scrambled siRNA (open columns) or siAC8 (filled columns) and expressing ANO1 and vector (black) or E-Syt1 (red) and treated with F/I (green). The results are from 3 independent experiments. G Current was measured with cells treated with scrambled (open columns) or siAC6 (filled columns) and expressing ANO1 and E-Syt1 + IRBIT (black) and treated with F/I (red). The results are from 4 independent experiments. H – J The Ca 2+ dependence of ANO1 in the presence of vector (black) stimulated with F/I (green), E-Syt1 (red) stimulated with F/I (turquoise), E-Syt1 + IRBIT (blue) stimulated with F/I (purple) ( H ), the Vmax ( I ) and apparent Km for Ca 2+ ( J ) were obtained from 5 independent experiments. Currents in ( A – D , F , G ) were measured with 0.3 µM Ca 2+ . All results are shown as mean ± s.e.m.

Article Snippet: The perfusate was switched to a Cl - -free solution by replacing Cl - in bath solution with NO 3 - , and after determining the baseline of MQAE fluorescence, the acini were stimulated with carbachol in the presence and absence of the ANO1 inhibitor Ani9 (5 μM, Cayman Chemical).

Techniques: Expressing, Transfection, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca 2+

doi: 10.1038/s41467-025-58682-w

Figure Lengend Snippet: A Current was measured in 4 independent experiments in the presence of 0.3 (open columns) or 10 µM pipette Ca 2+ (closed symbols/open and filled columns) in cells expressing ANO1 (black) and E-Syt2 (red) treated with F/I (green). In all experiments with the filled columns, the cells were also transfected with IRBIT. B Effect of E-Syt2 and F/I on E-Syt2-ANO1 Co-IP and on surface ANO1 expression. This is one of 3 similar experiments. C Current was measured in 3 independent experiments in cells treated with scrambled siRNA (black) or siE-Syt2 (all others) and expressing ANO1 (black, blue), IRBIT (green, red), and treated with F/I (filled columns) . D – F The Ca 2+ dependence of ANO1 in 4 independent experiments in the presence of vector (black), and in 5 independent experiments with E-Syt2 (red) and stimulated with F/I (green), E-Syt2+IRBIT (blue) and stimulated with F/I (purple) ( D ), the Vmax ( E ) and apparent Km for Ca 2+ ( F ). G Current was measured in 5 (or 4 with S221D) independent experiments with cells expressing ANO1 (blue), ANO1(S221A) (green) or ANO1(S221D) (red) and E-Syt2 and treated with F/I (filled columns) . H TIRF was used in 6 independent experiments to measure ANO1 (open symbols and columns), ANO1(S221A) (close symbols/open columns), and ANO1(S221D) (filled columns) puncta in the presence of vector (black) and IRBIT (green) and stimulated with F/I (blue, red). I – K The Ca 2+ dependence of ANO1 (black, blue, the same data as in Fig. ) and ANO1(S221D) (red, green) and stimulated with F/I (blue, green) ( I ), the Vmax ( J ) and apparent Km for Ca 2+ ( K ) were obtained from 3 independent experiments. L ANO1 current was measured in 4 independent experiments (black) with E-Syt2 (green) and stimulated with F/I (red) in cells that, in addition, express VAPA (close symbols/open columns), and in the filled columns IRBIT (black), IRBIT + VAPA (light green) stimulated with F/I (red) and E-Syt2 + VAPA + IRBIT (dark green) stimulated with F/I (dark red). Unless otherwise indicated, currents were measured with 0.3 µM Ca 2+ . All results are shown as mean ± s.e.m.

Article Snippet: The perfusate was switched to a Cl - -free solution by replacing Cl - in bath solution with NO 3 - , and after determining the baseline of MQAE fluorescence, the acini were stimulated with carbachol in the presence and absence of the ANO1 inhibitor Ani9 (5 μM, Cayman Chemical).

Techniques: Transferring, Expressing, Transfection, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca 2+

doi: 10.1038/s41467-025-58682-w

Figure Lengend Snippet: All experiments in Fig. 6 were conducted with STIM1 −/− cells. A , B ANO1 current (black) was measured in 5 independent experiments in the presence of IRBIT (green), E-Syt1 (blue), IRBIT + E-Syt1 (red), and stimulation with F/I (filled columns). C Surface ANO1 (black) was measured in 4 independent experiments in the presence of IRBIT (red) or IRBIT + E-Syt1 (green) in cells stimulation with F/I (filled columns). D Current was measured in 4 independent experiments in cells expressing ANO1 (black) and VAPA (green) and stimulated with F/I (red) and IRBIT (filled columns). E Surface ANO1 (black) was measured in 5 independent experiments in the presence of IRBIT (red), VAPA (green), or IRBIT + VAPA (blue). F The ANO1-YFP and IRBIT-CFP FRET was measured in 7 independent experiments in cells expressing ANO1 (black) and E-Syt1 (red), E-Syt2 (green) or VAPA (blue). G – I The Ca 2+ dependence of the ANO1 in the presence of vector (black), IRBIT (green), and stimulated with F/I (red) ( G ), the Vmax ( H ) and apparent Km for Ca 2+ ( I ) were obtained from 4 independent experiments. J ANO1 current was measured in 5 independent experiments with vector (black) and E-Syt2 (green) and treated with F/I (red) and in the presence of IRBIT (filled columns). K – M The Ca 2+ dependence of the ANO1 in the presence of vector (black), E-Syt2 (red) and stimulated with F/I (green), E-Syt2 + IRBIT (blue) and stimulated with F/I (purple) ( L ), the Vmax ( M ) and apparent Km for Ca 2+ ( N ) were obtained from 4 independent experiments. Unless otherwise indicated, currents were measured with 0.3 µM Ca 2+ . All results are shown as mean ± s.e.m.

Article Snippet: The perfusate was switched to a Cl - -free solution by replacing Cl - in bath solution with NO 3 - , and after determining the baseline of MQAE fluorescence, the acini were stimulated with carbachol in the presence and absence of the ANO1 inhibitor Ani9 (5 μM, Cayman Chemical).

Techniques: Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca 2+

doi: 10.1038/s41467-025-58682-w

Figure Lengend Snippet: A Cells treated with scrambled siRNA (open columns), siAC1 (filled columns), siAC3 (dotted columns), siAC8 (diagonal strips), or siAC6 (horizontal strips), where used to measure ANO1 current (black) in the presence of IRBIT (green) and stimulated with F/I (red). Results are from 3 independent experiments. B Current was measured in 3 independent experiments in cells transfected with vector (open columns) or AC8 (filled columns) expressing ANO1 (black, blue), IRBIT (red), and stimulated with F/I (Green and Blue). C Current was measured in 4 independent experiments at 0.3 (open columns) or 10 µM Ca 2+ (close columns) in cells transfected with ANO1 and vector (black) or AC6 (red) and stimulated with F/I (blue, green). D The Ca 2+ -dependent current was measured in 3 independent experiments at 1 µM Ca 2+ in cells that were treated with scrambled siRNA (black), siAC1 (brown), siAC3 (blue), siAC6 (green), siAC3 + siAC6 (red) or siAC1 + siAC6 (orange) and expressing ANO1 (open columns) and E-Syt2 (close columns). Cells were unstimulated (open symbols) or were stimulated with F/I (close symbols). E – G The Ca 2+ dependence of the ANO1 was measured in 5 independent experiments in cells treated with scrambled (SCR) siRNA (gray) and stimulated with F/I (purple), E-Syt2 (orange), and stimulated with F/I (dark yellow). Cells were treated with siAC6 (black) and stimulated with F/I (red), E-Syt2 (blue), and stimulated with F/I (green) ( E ), the Vmax ( F ), and apparent Km for Ca 2+ ( G ). H Current was measured in 3 independent experiments at 10 µM Ca 2+ in STIM1 −/− cells treated with scrambled (open columns) or siAC6 (filled columns) expressing ANO1 (black) and E-Syt2 (red) and stimulated with F/I (green). I – K The Ca 2+ dependence of the ANO1 was measured in 3 independent experiments in STIM1 −/− cells treated with scrambled (SCR) siRNA, expressing E-Syt2 (green), and stimulated with F/I (dark yellow). Cells were treated with siAC6 (blue) and stimulated with F/I (light blue) expressing E-Syt2 (red) and stimulated with F/I (purple) ( I ), with the Vmax ( J ) and apparent Km for Ca 2+ ( K ). All results are shown as mean ± s.e.m.

Article Snippet: The perfusate was switched to a Cl - -free solution by replacing Cl - in bath solution with NO 3 - , and after determining the baseline of MQAE fluorescence, the acini were stimulated with carbachol in the presence and absence of the ANO1 inhibitor Ani9 (5 μM, Cayman Chemical).

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca 2+

doi: 10.1038/s41467-025-58682-w

Figure Lengend Snippet: A – G Cells were treated with scrambled siRNA (Black), siAKAP3 (green), siAKAP5 (blue) or siAKAP11 (red) and were used to measure ( A ), ANO1 current in ( n = 4) (number of independent experiments in parenthesis). B ANO1 surface expression ( n = 3). C ANO1 current ( n = 6) with IRBIT and stimulated with F/I. D ANO1 current ( n = 5) with VAPA (striped columns) and stimulated with F/I (filled columns). The currents in the absence of VAPA are the same as in ( A ). E Surface ANO1 ( n = 3) when expressed with VAPA (striped columns) with cells stimulated with F/I (filled columns). F ANO1 current ( n = 4) with E-Syt1 (striped columns) and stimulated with F/I (filled columns). G ANO1 current ( n = 5) with E-Syt2 (striped columns) and stimulated with F/I (filled columns). H Current was measured ( n = 4) in cells expressing ANO1 and vector (open columns) or ANO1 + E-Syt2 (close columns) (black) and together with AKAP11 (green) and stimulated with F/I (red and blue). Unless otherwise indicated, all currents were measured with 0.3 µM Ca 2+ . All results are shown as mean ± s.e.m.

Article Snippet: The perfusate was switched to a Cl - -free solution by replacing Cl - in bath solution with NO 3 - , and after determining the baseline of MQAE fluorescence, the acini were stimulated with carbachol in the presence and absence of the ANO1 inhibitor Ani9 (5 μM, Cayman Chemical).

Techniques: Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca 2+

doi: 10.1038/s41467-025-58682-w

Figure Lengend Snippet: The Ca 2+ dependence of ANO1 current was measured in 4 independent experiments ( A – F ) in cells expressing: ( A – C ), E-Syt1 (blue), the SMP mutant E-Syt1 V169W/L308W (turquoise) and stimulated with F/I (green, red); ( D – F ) vector (black), E-Syt2 (blue), the SMP mutant E-Syt2 V197W/I337W (turquoise) and stimulated with F/I (green, red). Vmax ( B , E ) and apparent Km for Ca 2+ ( C , F ) are shown. The currents with E-Syt1 are from Fig. and with vector and E-Syt2 are from Fig. . G ANO1 current was measured in 3 independent experiments in a pipette solution containing 0.1 µM Ca 2+ and expressing ANO1 + E-Syt2 V197W/I337W and treated with SCR or E-Syt1 siRNA. H ANO1 current was measured in 3 independent experiments in a pipette solution containing 0.3 µM Ca 2+ and expressing the C2 domain mutants of E-Syt1 and E-Syt2, as indicated. (I–M) , Cl - current was measured in the immortalized human salivary glands acinar cell line NS-SV-AC. I – J The native Cl - current was measured in 5 (green) or 3 (all others) independent experiments at 0.3 (green, blue), 1 (red, black), and 30 µM Ca 2+ (brown) and in the presence of the ANO1 inhibitor 10 µM Ani9 (black, blue). K – M ANO1 Cl - current was measured in 3 independent experiments at 0.3 µM Ca 2+ in NS-SV-AC cells treated with ( K ), scrambled (SCR) siRNA (black), or siRNAs targeting VAPA (blue), E-Syt1 (red), E-Syt2 (green), IRBIT (purple) or STIM1 (orange); ( L ), AC8 (red) or AC6 (green); ( M ), AKAP3 (green), AKAP5 (blue), AKAP11 (red) and stimulated with F/I (filled columns). All results are shown as mean ± s.e.m.

Article Snippet: The perfusate was switched to a Cl - -free solution by replacing Cl - in bath solution with NO 3 - , and after determining the baseline of MQAE fluorescence, the acini were stimulated with carbachol in the presence and absence of the ANO1 inhibitor Ani9 (5 μM, Cayman Chemical).

Techniques: Expressing, Mutagenesis, Plasmid Preparation, Transferring