7. TMEM16A plays a part in flow-stimulated currents. fura 2-AM (TEF Labs, Austin, TX) in isotonic extracellular buffer formulated with (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 1 KH2PO4, 5 blood sugar, and 10 HEPES (pH 7.4) supplemented with 0.01% Pluronic F-127 for 30 min at 22C. In chosen research, EGTA (2 mM) was utilized to eliminate Ca2+ in the shower and perfusing solutions. The coverslip was put into the perfusion chamber in the stage of the inverted fluorescence microscope (Nikon TE2000), as well as the outflow and inflow slots had been mounted on the syringe pump. Adjustments in [Ca2+]we had been assessed at excitation wavelength of 340 nm for Ca2+-destined fura 2-AM and 380 nm for Ca2+-free of Menbutone charge fura 2-AM at emission wavelength of 510 nm. After subtraction of history fluorescence, [Ca2+]i was computed based on the Grynkiewicz formula (44): [Ca2+]i (nM) = = 101), email address details are reported as current thickness (pA/pF) to normalize for distinctions in cell size (13). CFTR and TMEM16A silencing. TMEM16A was suppressed by particular anti-TMEM16A little interfering RNA (siRNA; TMEM16A-HSS123904), as defined in our prior research (9). Quickly, 25-nucleotide siRNAs had been designed and synthesized by Invitrogen [AAG UUA GUG AGG UAG GCU GGG AAC C (antisense) and GGU UCC CAG CCU ACC UCA CUA ACU U (feeling)] and transfected using FuGENE (5 g/100 l). Noncoding Stealth RNAi (moderate guanine-cytosine duplex, Invitrogen) was employed in control (mock) transfections. Likewise, CFTR was suppressed by particular anti-CFTR siRNA (catalog no. 4392421, Lifestyle Technology). BLOCK-iT Fluorescent Oligo (catalog no. 2013, Invitrogen) was utilized to optimize transfection circumstances and to go for transfected cells for entire cell patch-clamp documenting. Entire cell patch-clamp tests had been performed 24C48 h after transfection. Transfection performance and the amount of TMEM16A and CFTR silencing had been measured on the message level by real-time PCR with the proteins level by Traditional western blot evaluation (9). Reagents. The CFTR inhibitors CFTR(inh)-172 and malic hydrazide (MalH) had been kind presents from Drs. Nitin Sonawane and Alan Verkman (School of California, SAN FRANCISCO BAY AREA, CA). Anti-CFTR (clone M3A7) monoclonal antibody (catalog no. 05-583) was purchased from Millipore. All the reagents had been extracted from Sigma-Aldrich (St. Louis, MO). Figures. Beliefs are means SE, with representing the real variety of lifestyle plates or repetitions for every assay. Statistical evaluation included Fisher’s matched and unpaired 0.05 was considered to be significant statistically. RESULTS Stream (shear) activates membrane Cl? currents. To characterize the pharmacological and biophysical properties of membrane Cl? currents in response to shear, entire cell patch-clamp research had been performed in one Mz-ChA-1 and H69 cells and MSC and MLC in the presence or absence of defined shear. Representative traces of a Mz-ChA-1 cell and a H669 cell are shown in Fig. 1. Under basal conditions with standard intra- and extracellular buffers, Cl? current was small (?1.9 0.5 pA/pF). Exposure to flow (shear = 0.24 dyn/cm2) resulted in activation of currents within 95 17 s, increasing current density to ?18.0 4.0 pA/pF at ?80 mV ( 0.001, = 13 for Mz-ChA-1 cells; 0.05, = 4 for H69 cells). The currents were sustained for.Whole cell currents were measured during basal conditions and during exposure to flow of isotonic extracellular buffer. Cl? currents mediated by TMEM16A. Identification of this novel mechanosensitive secretory pathway provides new insight into bile formation and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders. is usually chamber height (cm), and is chamber width (cm). Ca2+ imaging. Cells were cultured for 48 h on 15-mm glass coverslips and then loaded with 2.5 g/ml fura 2-AM (TEF Labs, Austin, TX) in isotonic extracellular buffer made up of (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 1 KH2PO4, 5 glucose, and 10 HEPES (pH 7.4) supplemented with 0.01% Pluronic F-127 for 30 min at 22C. In selected studies, EGTA (2 mM) was used to remove Ca2+ from the bath and perfusing solutions. The coverslip was placed in the perfusion chamber around the stage of an inverted fluorescence microscope (Nikon TE2000), and the inflow and outflow ports were attached to the syringe pump. Changes in [Ca2+]i were measured at excitation wavelength of 340 nm for Ca2+-bound fura 2-AM and 380 nm for Ca2+-free fura 2-AM at emission wavelength of 510 nm. After subtraction of background fluorescence, [Ca2+]i was calculated according to the Grynkiewicz equation (44): [Ca2+]i (nM) = = 101), results are reported as current density (pA/pF) to normalize for differences in cell size (13). TMEM16A and CFTR silencing. TMEM16A was suppressed by specific anti-TMEM16A small interfering RNA (siRNA; TMEM16A-HSS123904), as described in our previous studies (9). Briefly, 25-nucleotide siRNAs were N-Shc designed and synthesized by Invitrogen [AAG UUA GUG AGG UAG GCU GGG AAC C (antisense) and GGU UCC CAG CCU ACC UCA CUA ACU U (sense)] and transfected using FuGENE (5 g/100 l). Noncoding Stealth RNAi (medium guanine-cytosine duplex, Invitrogen) was utilized in control (mock) transfections. Similarly, CFTR was suppressed by specific anti-CFTR siRNA (catalog no. 4392421, Life Technologies). BLOCK-iT Fluorescent Oligo (catalog no. 2013, Invitrogen) was used to optimize transfection conditions and to select transfected cells for whole cell patch-clamp recording. Whole cell patch-clamp experiments were performed 24C48 h after transfection. Transfection efficiency and the degree of TMEM16A and CFTR silencing were measured at the message level by real-time PCR and at the protein level by Western blot analysis (9). Reagents. The CFTR inhibitors CFTR(inh)-172 and malic hydrazide (MalH) were kind gifts from Drs. Nitin Sonawane and Alan Verkman (University of California, San Francisco, CA). Anti-CFTR (clone M3A7) monoclonal antibody (catalog no. 05-583) was purchased from Millipore. All other reagents were obtained from Sigma-Aldrich (St. Louis, MO). Statistics. Values are means SE, with representing the number of culture plates or repetitions for each assay. Statistical analysis included Fisher’s paired and unpaired 0.05 was considered to be statistically significant. RESULTS Flow (shear) activates membrane Cl? currents. To characterize the biophysical and pharmacological properties of membrane Cl? currents in response to shear, whole cell patch-clamp studies were performed in single Mz-ChA-1 and H69 cells and MSC and MLC in the presence or absence of defined shear. Representative traces of a Mz-ChA-1 cell and a H669 cell are shown in Fig. 1. Under Menbutone basal conditions with standard intra- and extracellular buffers, Cl? current was small (?1.9 0.5 pA/pF). Exposure to flow (shear = 0.24 dyn/cm2) resulted in activation of currents within 95 17 s, increasing current density to ?18.0 4.0 pA/pF at ?80 mV ( 0.001, = 13 for Mz-ChA-1 cells; 0.05, = 4 for H69 cells). The currents were sustained for the duration of flow exposure and were fully reversible within 5 min of flow cessation. Interestingly, currents exhibited two distinct patterns. In the majority (85%) of studies, the currents exhibited reversal near 0 mV [Cl? reversal (equilibrium) potential], outward rectification, and time-dependent activation at depolarizing potentials above +60 mV (Fig. 1), characteristics associated with Ca2+-activated Cl? currents previously described in these cells (9, 16). However, in a minority (15%) of studies, currents exhibited time-dependent inactivation at positive depolarizing potentials above +60 mV (Fig. 2). In some studies, currents with both types of biophysical properties were Menbutone observed in the same cell (5 of 35). Inclusion of MgATP2? in the patch pipette increased (from 15% to 38%) the relative percentage of the currents displaying time-dependent.Masyuk AI, Masyuk TV, Splinter PL, Huang BQ, Stroope AJ, LaRusso NF. Cholangiocyte cilia detect changes in luminal fluid flow and transmit them into intracellular Ca2+ and cAMP signaling. Gastroenterology 131: 911C 920, 2006 [PMC free article] [PubMed] [Google Scholar] 36. Furthermore, activation of TMEM16A by flow is dependent on PKC through a process involving extracellular ATP, binding purinergic P2 receptors, and increases in intracellular Ca2+ concentration. These studies represent the initial characterization of mechanosensitive Cl? currents mediated by TMEM16A. Identification of this novel mechanosensitive secretory pathway provides new insight into bile formation and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders. is usually chamber height Menbutone (cm), and is chamber width (cm). Ca2+ imaging. Cells were cultured for 48 h on 15-mm glass coverslips and then loaded with 2.5 g/ml fura 2-AM (TEF Labs, Austin, TX) in isotonic extracellular buffer made up of (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 1 KH2PO4, 5 glucose, and 10 HEPES (pH 7.4) supplemented with 0.01% Pluronic F-127 for 30 min at 22C. In selected studies, EGTA (2 mM) was used to remove Ca2+ from the bath and perfusing solutions. The coverslip was placed in the perfusion chamber around the stage of an inverted fluorescence microscope (Nikon TE2000), and the inflow and outflow ports were attached to the syringe pump. Changes in [Ca2+]i were measured at excitation wavelength of 340 nm for Ca2+-bound fura 2-AM and 380 nm for Ca2+-free fura 2-AM at emission wavelength of 510 nm. After subtraction of background fluorescence, [Ca2+]i was calculated according to the Grynkiewicz equation (44): [Ca2+]i (nM) = = 101), results are reported as current density (pA/pF) to normalize for differences in cell size (13). TMEM16A and CFTR silencing. TMEM16A was suppressed by specific anti-TMEM16A small interfering RNA (siRNA; TMEM16A-HSS123904), as described in our previous studies (9). Briefly, 25-nucleotide siRNAs were designed and synthesized by Invitrogen [AAG UUA GUG AGG UAG GCU GGG AAC C (antisense) and GGU UCC CAG CCU ACC UCA CUA ACU U (sense)] and transfected using FuGENE (5 g/100 l). Noncoding Stealth RNAi (medium guanine-cytosine duplex, Invitrogen) was utilized in control (mock) transfections. Similarly, CFTR was suppressed by specific anti-CFTR siRNA (catalog no. 4392421, Life Technologies). BLOCK-iT Fluorescent Oligo (catalog no. 2013, Invitrogen) was used to optimize transfection conditions and to select transfected cells for whole cell patch-clamp recording. Whole cell patch-clamp experiments were performed 24C48 h after transfection. Transfection efficiency and the degree of TMEM16A and CFTR silencing were measured at the message level by real-time PCR and at the protein level by Western blot analysis (9). Reagents. The CFTR inhibitors CFTR(inh)-172 and malic hydrazide (MalH) were kind gifts from Drs. Nitin Sonawane and Alan Verkman (University of California, San Francisco, CA). Anti-CFTR (clone M3A7) monoclonal antibody (catalog no. 05-583) was purchased from Millipore. All other reagents were obtained from Sigma-Aldrich (St. Louis, MO). Statistics. Values are means SE, with representing the number of culture plates or repetitions for each assay. Statistical analysis included Fisher’s paired and unpaired 0.05 was considered to be statistically significant. RESULTS Flow (shear) activates membrane Cl? currents. To characterize the biophysical and pharmacological properties of membrane Cl? currents in response to shear, whole cell patch-clamp studies were performed in single Mz-ChA-1 and H69 cells and MSC and MLC in the presence or absence of defined shear. Representative traces of a Mz-ChA-1 cell and a H669 cell are shown in Fig. 1. Under basal conditions with standard intra- and extracellular buffers, Cl? current was small (?1.9 0.5 pA/pF). Exposure to flow (shear = 0.24 dyn/cm2) resulted in activation of currents within 95 17 s, increasing current density to ?18.0 4.0 pA/pF at ?80 mV ( 0.001, = 13 for Mz-ChA-1 cells; 0.05, = 4 for H69 cells). The currents were sustained for the duration of flow exposure and were fully reversible within 5 min of flow cessation. Interestingly, currents demonstrated two distinct patterns. In the majority (85%) of studies, the currents exhibited reversal near 0 mV [Cl? reversal (equilibrium) potential], outward rectification, and time-dependent activation at depolarizing potentials above +60 mV (Fig. 1), characteristics associated with Ca2+-activated Cl? currents previously described in these cells (9, 16). However, in a minority (15%) of studies, currents demonstrated time-dependent inactivation at positive depolarizing potentials above +60 mV (Fig. 2). In some studies, currents with both types of biophysical properties were observed in the same cell (5 of 35). Inclusion of MgATP2? in the patch pipette increased (from 15% to 38%) the relative percentage of the currents displaying time-dependent inactivation. Therefore, to minimize the currents demonstrating time-dependent inactivation, the majority of studies were performed without additional MgATP?2 in the pipette. Open in a separate window Fig. 1. Characterization.In contrast, flow-stimulated ATP release is regulated by PKC (45). as the operative channel. Furthermore, activation of TMEM16A by flow is dependent on PKC through a process involving extracellular ATP, binding purinergic P2 receptors, and increases in intracellular Ca2+ concentration. These studies represent the initial characterization of mechanosensitive Cl? currents mediated by TMEM16A. Identification of this novel mechanosensitive secretory pathway provides new insight into bile formation and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders. is chamber height (cm), and is chamber width (cm). Ca2+ imaging. Cells were cultured for 48 h on 15-mm glass coverslips and then loaded with 2.5 g/ml fura 2-AM (TEF Labs, Austin, TX) in isotonic extracellular buffer containing (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 1 KH2PO4, 5 glucose, and 10 HEPES (pH 7.4) supplemented with 0.01% Pluronic F-127 for 30 min at 22C. In selected studies, EGTA (2 mM) was used to remove Ca2+ from the bath and perfusing solutions. The coverslip was placed in the perfusion chamber on the stage of an inverted fluorescence microscope (Nikon TE2000), and the inflow and outflow ports were attached to the syringe pump. Changes in [Ca2+]i were measured at excitation wavelength of 340 nm for Ca2+-bound fura 2-AM and 380 nm for Ca2+-free fura 2-AM at emission wavelength of 510 nm. After subtraction of background fluorescence, [Ca2+]i was calculated according to the Grynkiewicz equation (44): [Ca2+]i (nM) = = 101), results are reported as current density (pA/pF) to normalize for differences in cell size (13). TMEM16A and CFTR silencing. TMEM16A was suppressed by specific anti-TMEM16A small interfering RNA (siRNA; TMEM16A-HSS123904), as described in our previous studies (9). Briefly, 25-nucleotide siRNAs were designed and synthesized by Invitrogen [AAG UUA GUG AGG UAG GCU GGG AAC C (antisense) and GGU UCC CAG CCU ACC UCA CUA ACU U (sense)] and transfected using FuGENE (5 g/100 l). Noncoding Stealth RNAi (medium guanine-cytosine duplex, Invitrogen) was utilized in control (mock) transfections. Similarly, CFTR was suppressed by specific anti-CFTR siRNA (catalog no. 4392421, Life Technologies). BLOCK-iT Fluorescent Oligo (catalog no. 2013, Invitrogen) was used to optimize transfection conditions and to select transfected cells for whole cell patch-clamp recording. Whole cell patch-clamp experiments were performed 24C48 h after transfection. Transfection efficiency and the degree of TMEM16A and CFTR silencing were measured at the message level by real-time PCR and at the protein level by Western blot analysis (9). Reagents. The CFTR inhibitors CFTR(inh)-172 and malic hydrazide (MalH) were kind gifts from Drs. Nitin Sonawane and Alan Verkman (University of California, San Francisco, CA). Anti-CFTR (clone M3A7) monoclonal antibody (catalog no. 05-583) was purchased from Millipore. All other reagents were obtained from Sigma-Aldrich (St. Louis, MO). Statistics. Values are means SE, with representing the number of culture plates or repetitions for each assay. Statistical analysis included Fisher’s paired and unpaired 0.05 was considered to be statistically significant. RESULTS Flow (shear) activates membrane Cl? currents. To characterize the biophysical and pharmacological properties of membrane Cl? currents in response to shear, whole cell patch-clamp studies were performed in single Mz-ChA-1 and H69 cells and MSC and MLC in the presence or absence of defined shear. Representative traces of a Mz-ChA-1 cell and a H669 cell are shown in Fig. 1. Under basal conditions with standard intra- and extracellular buffers, Cl? current was small (?1.9 0.5 pA/pF). Exposure to flow (shear = 0.24 dyn/cm2) resulted in activation of currents within 95 17 s, increasing current density to ?18.0 4.0 pA/pF at ?80 mV ( 0.001, = Menbutone 13 for Mz-ChA-1 cells; 0.05, = 4 for H69 cells). The currents were sustained for the duration of flow exposure and were fully reversible within 5 min of flow cessation. Interestingly, currents demonstrated two distinct patterns. In.