Journal:

Article Title: Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current

doi: 10.1113/jphysiol.2003.058032

Figure Lengend Snippet: A, representative currents in CLC-3−/−versus CLC-3+/+ mouse aorta smooth muscle cells were recorded at a minimum (Basal) and peak steady-state current level in the presence of CaMKII and 1 μm carbachol ± 1 μm Myr-AIP. Autonomously active CaMKII (10 μg ml−1) was introduced into the cell via the patch pipette. Carbachol was perfused onto the cells following basal recording. Cells treated with Myr-AIP were incubated prior to recording for 10 min. B, current–voltage relationship for currents recorded in A. C, summary of CaMKII-mediated activation in CLC-3−/−versus CLC-3+/+ mouse aorta smooth muscle cells. D, summary of carbachol-mediated activation in CLC-3−/−versus CLC-3+/+ mouse aorta smooth muscle cells. Data are expressed as mean ±s.e.m., with number of cells examined given in parentheses above each bar. E, protein obtained from homogenates of aortae or cultured aortic VSM cells used to identify native CLC-3 protein. Immunoblots confirm the depletion of CLC-3 protein in CLC-3−/− lanes (CLC-3, 90 kDa).

Article Snippet: A 100 μ m stock solution of the membrane-permeant CaMKII specific inhibitor Myr-AIP was diluted to 1 μ m with bath solution (Biomol Research Laboratories, Inc., PA, USA) ( Ishida et al. 1995 , 1998 ).

Techniques: Transferring, Incubation, Activation Assay, Cell Culture, Western Blot

Journal:

Article Title: Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current

doi: 10.1113/jphysiol.2003.058032

Figure Lengend Snippet: A, membrane topology of CLC-3 indicating the location of the Flag epitope, GST fusion constructs, and antibody recognition sites. Membrane topology was adapted from crystal structures of two bacterial CLC channels, StCLC and EcCLC (Dutzler et al. 2002). hCLC-3, StCLC, and EcCLC sequences were aligned using ClustalW (European Bioinformatics Institute) to determine regions of similarity. GST fusion proteins were constructed with the N- (1M to 122L) or C- (661R to 818N) terminal regions of human long form CLC-3. B, in vitro phosphorylation of CLC-3 by CaMKII. The cell lysate of tsA cells stably transfected with hCLC-3 was immunoprecipitated with α-hCLC-3730–744. The precipitated CLC-3 or rabbit synapsin was phosphorylated with CaMKII in the presence of [γ-32P]ATP, and resolved on SDS-PAGE. The resulting gel bands were detected using autoradiography (arrows: CLC-3, 120 kDa (glycosylated), synapsin (Syn) ∼84 kDa. C, in vitro phosphorylation of GST CLC-3 fusion proteins by CaMKII. The GST CLC-3 or rabbit synapsin was phosphorylated with CaMKII in the presence of [γ-32P]ATP, and resolved on SDS-PAGE. The resulting gel bands were detected using autoradiography (phosphorylation) and Coomassie blue to detect the presence of protein. Similar results were obtained in three experiments.

Article Snippet: A 100 μ m stock solution of the membrane-permeant CaMKII specific inhibitor Myr-AIP was diluted to 1 μ m with bath solution (Biomol Research Laboratories, Inc., PA, USA) ( Ishida et al. 1995 , 1998 ).

Techniques: Membrane, FLAG-tag, Construct, In Vitro, Phospho-proteomics, Stable Transfection, Transfection, Immunoprecipitation, SDS Page, Autoradiography

Journal:

Article Title: Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current

doi: 10.1113/jphysiol.2003.058032

Figure Lengend Snippet: A: upper panels, HT29 cells expressing native CLC-3 were permeabilized and localization was visualized with α-hCLC-3730–744. PBS-treated cells (Control) show a perinuclear distribution of CLC-3. Cells treated with 10 μm A23187, a Ca2+ ionophore, showed a diffuse cytosolic distribution of CLC-3. Cells treated with 1 μm of the specific CaMKII inhibitor, autocamtide-2 inhibitory peptide (AIP), prior to treatment with A23187, showed the same diffuse distribution as those treated with A23187 alone; lower panels, HT29 cells were transfected with Flag-CLC-3, and in these non-permeabilized cells there is no apparent difference in fluorescence labelling following intracellular Ca2+ elevation (same treatment conditions as in non-transfected cells). Similar results were seen in three experiments.B, representative traces of membrane capacitance (Cm) and membrane conductance (Gm) with (CaMKII) or without (Basal) autonomous CaMKII in the pipette. The holding potential was −5 mV. The arrow indicates initiation of whole-cell configuration. C, summary of changes in capacitance and conductance in the presence and absence Trafficking of CLC-3 is not CaMKII dependent and is not necessary for kinase-dependent conductance increase of CaMKII. The percentage change is relative to baseline levels following initiation of whole-cell configuration. D, immunoblot of CLC-3 immunoprecipitated (IP) with α-hCLC-3730–744 from HT29 cells or CLC-3 stably transfected tsA cells, blotted with α-hCLC-359–74 (molecular mass, ∼120 kDa, glycosylated). No CLC-3 protein was detected in HT29 supernatant (Sup) lane. E, summary of CaMKII-activated Cl− current densities in HT29 cells, with (CaMKII ± AIP) or without (Basal) autonomous CaMKII in the pipette. AIP (1 μm) was included in the pipette solution where indicated. *Significant difference (P < 0.01). Data are expressed as mean ±s.e.m., with number of cells examined given in parentheses above each bar.

Article Snippet: A 100 μ m stock solution of the membrane-permeant CaMKII specific inhibitor Myr-AIP was diluted to 1 μ m with bath solution (Biomol Research Laboratories, Inc., PA, USA) ( Ishida et al. 1995 , 1998 ).

Techniques: Expressing, Control, Transfection, Fluorescence, Membrane, Transferring, Western Blot, Immunoprecipitation, Stable Transfection

Journal:

Article Title: Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current

doi: 10.1113/jphysiol.2003.058032

Figure Lengend Snippet: A, representative currents in transfected wt CLC-3 versus S109A CLC-3 tsA cells were recorded at a minimum (Basal) and after they reached steady state in the presence of CaMKII. Autonomously active CaMKII was introduced into the cell via the patch pipette. CLC-3 (wt or S109A) was transfected into tsA cells 48 h prior to experiment. Green fluorescent protein (GFP) was cotransfected to identify positive cells. B, current–voltage relationship for A. C, summary of CaMKII-mediated activation in tsA cells transiently transfected with wt CLC-3 versus S109A CLC-3 or mock-transfected cells. Data are expressed as mean ±s.e.m., with number of cells examined given in parentheses above each bar. D, immunostaining of non-permeabilized tsA cells transfected with wt or S109A Flag-CLC-3. Plasma membrane localization is visualized after incubation with an anti-Flag antibody. E, representative time course of activation in three CLC-3 stably transfected tsA cells with (CaMKII) and without (Basal) CaMKII in the pipette, and 1 μm carbachol in the bath. Time zero for the CaMKII trace is initiation of whole-cell configuration; time zero for carbachol trace is initiation of carbachol superfusion into the bath.

Article Snippet: A 100 μ m stock solution of the membrane-permeant CaMKII specific inhibitor Myr-AIP was diluted to 1 μ m with bath solution (Biomol Research Laboratories, Inc., PA, USA) ( Ishida et al. 1995 , 1998 ).

Techniques: Transfection, Transferring, Activation Assay, Immunostaining, Clinical Proteomics, Membrane, Incubation, Stable Transfection