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    Olympus whole cell patch clamp recordings
    Safranal ( SFR ) and picrocrocin ( PICR ) selectively target the human TRPA 1 channel. A, Chemical structures of SFR , PICR and crocin ( CRO ). B, Concentration response curves of the calcium mobilization evoked by SFR and PICR in hTRPA 1 HEK 293 cells. Representative traces and pooled data of calcium response evoked by SFR , PICR and AITC in hTRPA 1 HEK 293 pre‐exposed to HC ‐030031 ( HC 03, 30 μmol/L) or its vehicle (‐) and in naïve HEK 293 cells. C, Representative traces and pooled data of <t>whole‐cell</t> <t>patch‐clamp</t> inward currents evoked by CRO and AITC (100 μmol/L) in hTRPA 1 HEK 293. D, Pooled data of calcium responses evoked by SFR and menthol in wild‐type (wt) and mutant (3C/K‐Q) hTRPA 1 HEK 293 transfected cells. E, Concentration response curves of the calcium mobilization evoked by SFR and PICR in IMR 90 cells. Representative traces and pooled data of calcium responses evoked by SFR , PICR and AITC pre‐exposed HC 03 (30 μmol/L) or its vehicle (‐) in IMR 90 cells. HC 03 does not affect the response evoked by the hPAR 2 ‐ AP (100 μmol/L). Veh is the vehicle of SFR , dash (‐) indicates the vehicle of HC 03. Data are mean ± SEM of n > 20 cells from 4 to 6 independent experiments (B, D, E) and n > 3 cells from 3 to 5 independent experiments (C). * P
    Whole Cell Patch Clamp Recordings, supplied by Olympus, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Molecular Devices LLC axopatch 200b amplifier
    Safranal ( SFR ) and picrocrocin ( PICR ) selectively target the human TRPA 1 channel. A, Chemical structures of SFR , PICR and crocin ( CRO ). B, Concentration response curves of the calcium mobilization evoked by SFR and PICR in hTRPA 1 HEK 293 cells. Representative traces and pooled data of calcium response evoked by SFR , PICR and AITC in hTRPA 1 HEK 293 pre‐exposed to HC ‐030031 ( HC 03, 30 μmol/L) or its vehicle (‐) and in naïve HEK 293 cells. C, Representative traces and pooled data of <t>whole‐cell</t> <t>patch‐clamp</t> inward currents evoked by CRO and AITC (100 μmol/L) in hTRPA 1 HEK 293. D, Pooled data of calcium responses evoked by SFR and menthol in wild‐type (wt) and mutant (3C/K‐Q) hTRPA 1 HEK 293 transfected cells. E, Concentration response curves of the calcium mobilization evoked by SFR and PICR in IMR 90 cells. Representative traces and pooled data of calcium responses evoked by SFR , PICR and AITC pre‐exposed HC 03 (30 μmol/L) or its vehicle (‐) in IMR 90 cells. HC 03 does not affect the response evoked by the hPAR 2 ‐ AP (100 μmol/L). Veh is the vehicle of SFR , dash (‐) indicates the vehicle of HC 03. Data are mean ± SEM of n > 20 cells from 4 to 6 independent experiments (B, D, E) and n > 3 cells from 3 to 5 independent experiments (C). * P
    Axopatch 200b Amplifier, supplied by Molecular Devices LLC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    HEKA Elektronik whole cell patch clamp recordings
    Paired <t>Recordings</t> Reveal that BAPTA Decreases Exocytosis and the Transient Block of Presynaptic Ca 2+ Currents (I Ca TB) in Mature Bullfrog Auditory Hair Cells (A1) Representative trace of paired <t>whole-cell</t> <t>voltage-clamp</t> recordings of presynaptic hair cell and a post-synaptic afferent fiber. The hair cell intracellular pipette solution included 10 mM BAPTA, and the external solution contained 25 mM bicarbonate (HCO 3 − ) as a pH buffer. The hair cell was depolarized from −60 to −30 mV for 20 ms after 10 s (black), 3 min (green), and 5 min (red) from the whole-cell break-in. The multiquantal EPSC had a large amplitude that correlates with the proton inhibition of the Ca 2+ currents. As 10 mM BAPTA dialyzed into the hair cell, the EPSC amplitude decreased, and the proton effect on the Ca 2+ current was removed (red trace), in contrast with recordings obtained 10 s after the whole-cell break-in (black). (A2) An expanded timescale of (A1) shows that 10 mM BAPTA delays the first EPSC event (green) and strongly decreases the EPSC amplitude. (B) The average EPSC charge of the later recordings ( > 4 min after the break-in, 2.0 ± 0.7 pC) was significantly decreased in comparison with the initial recordings (
    Whole Cell Patch Clamp Recordings, supplied by HEKA Elektronik, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Sutter Instrument whole cell patch clamp recordings
    Ca 2+ dependence of anion selectivity for TMEM16A is abolished by mutations of basic residues. ( A ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A in <t>whole-cell</t> <t>patch</t> <t>clamp</t> with 400 nM Ca 2+ in the pipette. Intracellular solution contained 140 mM NaCl, whereas extracellular ion concentrations are as indicated. ( B ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A with 1 mMCa 2+ in the pipette. ( C ) Permeability ratios are determined by solving the Goldman–Hodgkin–Katz voltage equation for each solution compared with chloride in the indicated concentrations of Ca 2+ . Permeability ratios for thiocyanate over chloride are shown for alanine mutants at ( D ) 400 nM Ca 2+ and ( E ) 1 mM Ca 2+ . N.S., not significant. *** P
    Whole Cell Patch Clamp Recordings, supplied by Sutter Instrument, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Safranal ( SFR ) and picrocrocin ( PICR ) selectively target the human TRPA 1 channel. A, Chemical structures of SFR , PICR and crocin ( CRO ). B, Concentration response curves of the calcium mobilization evoked by SFR and PICR in hTRPA 1 HEK 293 cells. Representative traces and pooled data of calcium response evoked by SFR , PICR and AITC in hTRPA 1 HEK 293 pre‐exposed to HC ‐030031 ( HC 03, 30 μmol/L) or its vehicle (‐) and in naïve HEK 293 cells. C, Representative traces and pooled data of whole‐cell patch‐clamp inward currents evoked by CRO and AITC (100 μmol/L) in hTRPA 1 HEK 293. D, Pooled data of calcium responses evoked by SFR and menthol in wild‐type (wt) and mutant (3C/K‐Q) hTRPA 1 HEK 293 transfected cells. E, Concentration response curves of the calcium mobilization evoked by SFR and PICR in IMR 90 cells. Representative traces and pooled data of calcium responses evoked by SFR , PICR and AITC pre‐exposed HC 03 (30 μmol/L) or its vehicle (‐) in IMR 90 cells. HC 03 does not affect the response evoked by the hPAR 2 ‐ AP (100 μmol/L). Veh is the vehicle of SFR , dash (‐) indicates the vehicle of HC 03. Data are mean ± SEM of n > 20 cells from 4 to 6 independent experiments (B, D, E) and n > 3 cells from 3 to 5 independent experiments (C). * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal, et al. TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal

    doi: 10.1111/jcmm.14099

    Figure Lengend Snippet: Safranal ( SFR ) and picrocrocin ( PICR ) selectively target the human TRPA 1 channel. A, Chemical structures of SFR , PICR and crocin ( CRO ). B, Concentration response curves of the calcium mobilization evoked by SFR and PICR in hTRPA 1 HEK 293 cells. Representative traces and pooled data of calcium response evoked by SFR , PICR and AITC in hTRPA 1 HEK 293 pre‐exposed to HC ‐030031 ( HC 03, 30 μmol/L) or its vehicle (‐) and in naïve HEK 293 cells. C, Representative traces and pooled data of whole‐cell patch‐clamp inward currents evoked by CRO and AITC (100 μmol/L) in hTRPA 1 HEK 293. D, Pooled data of calcium responses evoked by SFR and menthol in wild‐type (wt) and mutant (3C/K‐Q) hTRPA 1 HEK 293 transfected cells. E, Concentration response curves of the calcium mobilization evoked by SFR and PICR in IMR 90 cells. Representative traces and pooled data of calcium responses evoked by SFR , PICR and AITC pre‐exposed HC 03 (30 μmol/L) or its vehicle (‐) in IMR 90 cells. HC 03 does not affect the response evoked by the hPAR 2 ‐ AP (100 μmol/L). Veh is the vehicle of SFR , dash (‐) indicates the vehicle of HC 03. Data are mean ± SEM of n > 20 cells from 4 to 6 independent experiments (B, D, E) and n > 3 cells from 3 to 5 independent experiments (C). * P

    Article Snippet: For whole‐cell patch‐clamp recordings, coverslips with cells were transferred to a recording chamber (1 mL volume), mounted on the platform of an inverted microscope (Olympus CKX41) and superfused at a flow rate of 2 mL/min with a standard extracellular solution at pH 7.4 (adjusted with NaOH) containing (in mmol/L): 10 HEPES, 10 D‐glucose, 147 NaCl, 4 KCl, 1 MgCl2 , and 2 CaCl2 .

    Techniques: Concentration Assay, Patch Clamp, Mutagenesis, Transfection

    Safranal ( SFR ) causes desensitization. A, Representative traces and pooled data of whole‐cell patch‐clamp inward currents of the concentration dependant desensitization induced by SFR in response to AITC and KC l in hTRPA 1 HEK 293. B, Representative traces and pooled data of whole‐cell patch‐clamp inward currents of the desensitization induced by SFR in response to AITC and capsaicin ( CPS ) in rat DRG ( rDRG ) neurons. C, Pooled data of the desensitization induced by SFR in the contractile response evoked by SFR , AITC , CPS , GSK and carbachol ( CC h). D, Pooled data of the desensitization induced by SFR in CGRP ‐ LI release from rat spinal cord evoked by AITC , CPS and GSK . Veh is the vehicle of SFR . Data are mean ± SEM of n > 3 cells from 3‐5 independent experiments (A, B) and 4‐6 independent experiments (C, D). § P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal, et al. TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal

    doi: 10.1111/jcmm.14099

    Figure Lengend Snippet: Safranal ( SFR ) causes desensitization. A, Representative traces and pooled data of whole‐cell patch‐clamp inward currents of the concentration dependant desensitization induced by SFR in response to AITC and KC l in hTRPA 1 HEK 293. B, Representative traces and pooled data of whole‐cell patch‐clamp inward currents of the desensitization induced by SFR in response to AITC and capsaicin ( CPS ) in rat DRG ( rDRG ) neurons. C, Pooled data of the desensitization induced by SFR in the contractile response evoked by SFR , AITC , CPS , GSK and carbachol ( CC h). D, Pooled data of the desensitization induced by SFR in CGRP ‐ LI release from rat spinal cord evoked by AITC , CPS and GSK . Veh is the vehicle of SFR . Data are mean ± SEM of n > 3 cells from 3‐5 independent experiments (A, B) and 4‐6 independent experiments (C, D). § P

    Article Snippet: For whole‐cell patch‐clamp recordings, coverslips with cells were transferred to a recording chamber (1 mL volume), mounted on the platform of an inverted microscope (Olympus CKX41) and superfused at a flow rate of 2 mL/min with a standard extracellular solution at pH 7.4 (adjusted with NaOH) containing (in mmol/L): 10 HEPES, 10 D‐glucose, 147 NaCl, 4 KCl, 1 MgCl2 , and 2 CaCl2 .

    Techniques: Patch Clamp, Concentration Assay

    Safranal ( SFR ) selectively activates TRPA 1 in rodent primary sensory neurons. A, Concentration response curves of the calcium mobilization evoked by SFR and AITC in rat DRG ( rDRG ) neurons. Representative traces and pooled data of calcium response evoked by SFR , AITC and capsaicin ( CPS ) in rDRG neurons pre‐exposed to HC ‐030031 ( HC 03, 50 μmol/L), capsazepine ( CPZ ; 10 μmol/L), HC ‐067047 ( HC 06; 30 μmol/L) or their vehicles (‐). B, Representative traces and pooled data of whole‐cell patch‐clamp inward currents evoked by SFR , AITC and CPS in rDRG neurons. HC 03 does not affect the responses evoked by CPS . C, Representative traces and pooled data of the calcium responses evoked by SFR or AITC in mouse DRG ( mDRG ) neurons from Trpa1 +/+ and Trpa1 −/− mice. Veh is the vehicle of SFR . Dash (‐) indicates vehicles of the different treatments. Data are mean ± SEM of n > 20 cells from 4 to 6 independent experiments (A, C) and n > 3 cells from 3 to 5 independent experiments (B). * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal, et al. TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal

    doi: 10.1111/jcmm.14099

    Figure Lengend Snippet: Safranal ( SFR ) selectively activates TRPA 1 in rodent primary sensory neurons. A, Concentration response curves of the calcium mobilization evoked by SFR and AITC in rat DRG ( rDRG ) neurons. Representative traces and pooled data of calcium response evoked by SFR , AITC and capsaicin ( CPS ) in rDRG neurons pre‐exposed to HC ‐030031 ( HC 03, 50 μmol/L), capsazepine ( CPZ ; 10 μmol/L), HC ‐067047 ( HC 06; 30 μmol/L) or their vehicles (‐). B, Representative traces and pooled data of whole‐cell patch‐clamp inward currents evoked by SFR , AITC and CPS in rDRG neurons. HC 03 does not affect the responses evoked by CPS . C, Representative traces and pooled data of the calcium responses evoked by SFR or AITC in mouse DRG ( mDRG ) neurons from Trpa1 +/+ and Trpa1 −/− mice. Veh is the vehicle of SFR . Dash (‐) indicates vehicles of the different treatments. Data are mean ± SEM of n > 20 cells from 4 to 6 independent experiments (A, C) and n > 3 cells from 3 to 5 independent experiments (B). * P

    Article Snippet: For whole‐cell patch‐clamp recordings, coverslips with cells were transferred to a recording chamber (1 mL volume), mounted on the platform of an inverted microscope (Olympus CKX41) and superfused at a flow rate of 2 mL/min with a standard extracellular solution at pH 7.4 (adjusted with NaOH) containing (in mmol/L): 10 HEPES, 10 D‐glucose, 147 NaCl, 4 KCl, 1 MgCl2 , and 2 CaCl2 .

    Techniques: Concentration Assay, Patch Clamp, Mouse Assay

    Paired Recordings Reveal that BAPTA Decreases Exocytosis and the Transient Block of Presynaptic Ca 2+ Currents (I Ca TB) in Mature Bullfrog Auditory Hair Cells (A1) Representative trace of paired whole-cell voltage-clamp recordings of presynaptic hair cell and a post-synaptic afferent fiber. The hair cell intracellular pipette solution included 10 mM BAPTA, and the external solution contained 25 mM bicarbonate (HCO 3 − ) as a pH buffer. The hair cell was depolarized from −60 to −30 mV for 20 ms after 10 s (black), 3 min (green), and 5 min (red) from the whole-cell break-in. The multiquantal EPSC had a large amplitude that correlates with the proton inhibition of the Ca 2+ currents. As 10 mM BAPTA dialyzed into the hair cell, the EPSC amplitude decreased, and the proton effect on the Ca 2+ current was removed (red trace), in contrast with recordings obtained 10 s after the whole-cell break-in (black). (A2) An expanded timescale of (A1) shows that 10 mM BAPTA delays the first EPSC event (green) and strongly decreases the EPSC amplitude. (B) The average EPSC charge of the later recordings ( > 4 min after the break-in, 2.0 ± 0.7 pC) was significantly decreased in comparison with the initial recordings (

    Journal: Cell reports

    Article Title: Clustered Ca2+ Channels Are Blocked by Synaptic Vesicle Proton Release at Mammalian Auditory Ribbon Synapses

    doi: 10.1016/j.celrep.2018.11.072

    Figure Lengend Snippet: Paired Recordings Reveal that BAPTA Decreases Exocytosis and the Transient Block of Presynaptic Ca 2+ Currents (I Ca TB) in Mature Bullfrog Auditory Hair Cells (A1) Representative trace of paired whole-cell voltage-clamp recordings of presynaptic hair cell and a post-synaptic afferent fiber. The hair cell intracellular pipette solution included 10 mM BAPTA, and the external solution contained 25 mM bicarbonate (HCO 3 − ) as a pH buffer. The hair cell was depolarized from −60 to −30 mV for 20 ms after 10 s (black), 3 min (green), and 5 min (red) from the whole-cell break-in. The multiquantal EPSC had a large amplitude that correlates with the proton inhibition of the Ca 2+ currents. As 10 mM BAPTA dialyzed into the hair cell, the EPSC amplitude decreased, and the proton effect on the Ca 2+ current was removed (red trace), in contrast with recordings obtained 10 s after the whole-cell break-in (black). (A2) An expanded timescale of (A1) shows that 10 mM BAPTA delays the first EPSC event (green) and strongly decreases the EPSC amplitude. (B) The average EPSC charge of the later recordings ( > 4 min after the break-in, 2.0 ± 0.7 pC) was significantly decreased in comparison with the initial recordings (

    Article Snippet: Whole cell patch-clamp recordings were performed by using an EPC10 amplifier controlled by Patchmaster software (HEKA Elektronik, Germany).

    Techniques: Blocking Assay, Transferring, Mass Spectrometry, Inhibition

    Ca 2+ dependence of anion selectivity for TMEM16A is abolished by mutations of basic residues. ( A ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A in whole-cell patch clamp with 400 nM Ca 2+ in the pipette. Intracellular solution contained 140 mM NaCl, whereas extracellular ion concentrations are as indicated. ( B ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A with 1 mMCa 2+ in the pipette. ( C ) Permeability ratios are determined by solving the Goldman–Hodgkin–Katz voltage equation for each solution compared with chloride in the indicated concentrations of Ca 2+ . Permeability ratios for thiocyanate over chloride are shown for alanine mutants at ( D ) 400 nM Ca 2+ and ( E ) 1 mM Ca 2+ . N.S., not significant. *** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

    doi: 10.1073/pnas.1502291112

    Figure Lengend Snippet: Ca 2+ dependence of anion selectivity for TMEM16A is abolished by mutations of basic residues. ( A ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A in whole-cell patch clamp with 400 nM Ca 2+ in the pipette. Intracellular solution contained 140 mM NaCl, whereas extracellular ion concentrations are as indicated. ( B ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A with 1 mMCa 2+ in the pipette. ( C ) Permeability ratios are determined by solving the Goldman–Hodgkin–Katz voltage equation for each solution compared with chloride in the indicated concentrations of Ca 2+ . Permeability ratios for thiocyanate over chloride are shown for alanine mutants at ( D ) 400 nM Ca 2+ and ( E ) 1 mM Ca 2+ . N.S., not significant. *** P

    Article Snippet: Whole-cell patch-clamp recordings were performed using borosilicate capillary glass electrodes (Sutter Instrument) polished to a tip resistance of 3–5 MΩ.

    Techniques: Patch Clamp, Transferring, Permeability

    Alanine scan of putative pore-lining basic residues identifies five that significantly increase permeability ratios in bi-ionic condition. ( A ) Proposed topology of TMEM16A channel with basic residues within the putative pore region or previously implicated in permeation indicated at their predicted locations. ( B ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp in the constructs indicated. Color coding indicates extracellular ion species (all at 140 mM). Black, NaCl; blue, NaI; green, NaSCN; red, NaBr. Permeability ratios for all alanine mutants as determined by solving the Goldman–Hodgkin–Katz voltage equation for ( C ) bromide, ( D ) iodide, and ( E ) thiocyanate compared with chloride for each construct. Ratios are compared using one-way ANOVA followed by Tukey’s posthoc test. Statistical comparison of WT TMEM16A in whole-cell mode with other constructs is indicated with the top bar. ***Significant difference ( P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

    doi: 10.1073/pnas.1502291112

    Figure Lengend Snippet: Alanine scan of putative pore-lining basic residues identifies five that significantly increase permeability ratios in bi-ionic condition. ( A ) Proposed topology of TMEM16A channel with basic residues within the putative pore region or previously implicated in permeation indicated at their predicted locations. ( B ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp in the constructs indicated. Color coding indicates extracellular ion species (all at 140 mM). Black, NaCl; blue, NaI; green, NaSCN; red, NaBr. Permeability ratios for all alanine mutants as determined by solving the Goldman–Hodgkin–Katz voltage equation for ( C ) bromide, ( D ) iodide, and ( E ) thiocyanate compared with chloride for each construct. Ratios are compared using one-way ANOVA followed by Tukey’s posthoc test. Statistical comparison of WT TMEM16A in whole-cell mode with other constructs is indicated with the top bar. ***Significant difference ( P

    Article Snippet: Whole-cell patch-clamp recordings were performed using borosilicate capillary glass electrodes (Sutter Instrument) polished to a tip resistance of 3–5 MΩ.

    Techniques: Permeability, Patch Clamp, Construct

    Alanine mutants that alter anion selectivity also affect potency of pore blockers. ( A ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution with increasing concentrations of blocker NTTP as indicated. ( B ) Concentration–response curves are plotted for NTTP for TMEM16A and alanine mutants that alter anion selectivity. ( C ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution with increasing concentrations of blocker 1PBC as indicated. ( D ) Concentration–response curves are plotted for 1PBC for TMEM16A and alanine mutants that alter anion selectivity.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

    doi: 10.1073/pnas.1502291112

    Figure Lengend Snippet: Alanine mutants that alter anion selectivity also affect potency of pore blockers. ( A ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution with increasing concentrations of blocker NTTP as indicated. ( B ) Concentration–response curves are plotted for NTTP for TMEM16A and alanine mutants that alter anion selectivity. ( C ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution with increasing concentrations of blocker 1PBC as indicated. ( D ) Concentration–response curves are plotted for 1PBC for TMEM16A and alanine mutants that alter anion selectivity.

    Article Snippet: Whole-cell patch-clamp recordings were performed using borosilicate capillary glass electrodes (Sutter Instrument) polished to a tip resistance of 3–5 MΩ.

    Techniques: Patch Clamp, Concentration Assay

    Characterization of two pore blockers from a high-throughput small molecule screen. ( A ) Structure of TMEM16A inhibitor (PubChem SID 50085892) NTTP. ( B ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution (black trace) and bath solution with 30 μM NTTP (red trace). ( C ) Voltage dependence of block is calculated at positive potentials from the Woodhull equation, and effective block distance from the external face is shown as zδ . ( D ) Sample traces showing blocker onset and washout in ( Upper ) 140 mM NaCl solution and ( Lower ) low ionic solution containing 14 mM NaCl supplemented with sucrose. ( E ) Structure of TMEM16A inhibitor (PubChem SID 49642647) 1PBC. ( F ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution (black trace) and bath solution with 30 μM 1PBC (blue trace). ( G ) Voltage dependence of block is calculated at positive potentials from the Woodhull equation. ( H ) Sample traces showing blocker onset and washout when membrane potential is held at +60 mV in ( Upper ) 140 mM NaCl solution and ( Lower ) low ionic solution containing 14 mM NaCl supplemented with sucrose. ( I ) Time constants fit to traces in D for block and unblock on washout of NTTP. ( J ) Time constants from double exponentials fit to blocking time course for traces in I for 1PBC with either 140 or 14 mM NaCl. ( K ) Time constants from double exponentials fit to unblock time course for traces in I . K , Inset shows expanded view of comparison of fast time constants. *** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

    doi: 10.1073/pnas.1502291112

    Figure Lengend Snippet: Characterization of two pore blockers from a high-throughput small molecule screen. ( A ) Structure of TMEM16A inhibitor (PubChem SID 50085892) NTTP. ( B ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution (black trace) and bath solution with 30 μM NTTP (red trace). ( C ) Voltage dependence of block is calculated at positive potentials from the Woodhull equation, and effective block distance from the external face is shown as zδ . ( D ) Sample traces showing blocker onset and washout in ( Upper ) 140 mM NaCl solution and ( Lower ) low ionic solution containing 14 mM NaCl supplemented with sucrose. ( E ) Structure of TMEM16A inhibitor (PubChem SID 49642647) 1PBC. ( F ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution (black trace) and bath solution with 30 μM 1PBC (blue trace). ( G ) Voltage dependence of block is calculated at positive potentials from the Woodhull equation. ( H ) Sample traces showing blocker onset and washout when membrane potential is held at +60 mV in ( Upper ) 140 mM NaCl solution and ( Lower ) low ionic solution containing 14 mM NaCl supplemented with sucrose. ( I ) Time constants fit to traces in D for block and unblock on washout of NTTP. ( J ) Time constants from double exponentials fit to blocking time course for traces in I for 1PBC with either 140 or 14 mM NaCl. ( K ) Time constants from double exponentials fit to unblock time course for traces in I . K , Inset shows expanded view of comparison of fast time constants. *** P

    Article Snippet: Whole-cell patch-clamp recordings were performed using borosilicate capillary glass electrodes (Sutter Instrument) polished to a tip resistance of 3–5 MΩ.

    Techniques: High Throughput Screening Assay, Patch Clamp, Blocking Assay