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1.
Figure 7

Figure 7. Qualitative and quantitative analysis of ClC-1–ClC-5 transcripts in atria and brain. From: Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice.

A is a qualitative study. ClC-1 = 161 bp, ClC-2 = 193 bp, ClC-3 = 174 bp, ClC-4 = 174 bp, ClC-5 = 189 bp. NTC panel demonstrates that reactions lacking cDNA template did not amplify any products. Ladder bands indicate the molecular weight of amplicons and the sizes are indicated at the left side of panel A. B is a quantitative experiment that is measuring the ratio of expression of ClC channel transcripts of KO to that of WT mice. Therefore, if there is no expression (see, e.g. ClC-4) or no change in expression in KO compared to WT atria or brain tissues the ratio will be 1.

Shintaro Yamamoto-Mizuma, et al. J Physiol. 2004 June 1;557(Pt 2):439-456.
2.
Figure 4

Figure 4. Comparison of effects of intracellular dialysis of anti-ClC-3 Ab (A1–14 Ab) on VSOAC currents in atrial myocytes from Clcn3+/+ (WT) and Clcn3−/− (KO) mice. From: Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice.

A and B, time course of changes of VSOAC currents at +80 mV (•) and −80 mV (○) in atrial cells from WT (A) and KO (B) mice with intracellular dialysis of 1.5 μg ml−1 anti-ClC-3 antibody (A1–14 Ab). C and D, VSOAC currents in myocytes from WT mice (Ca and b) and from KO mice (Da and b) in isotonic and hypotonic solutions, respectively. Currents were recorded at the time points (a and b) indicated in A and B. E and F, mean IV relationships of WT (n = 10) and KO (n = 7) in isotonic and hypotonic solutions, respectively. G, mean IV relationships of hypotonic-activated Cl currents in myocytes from WT (•) and KO (○) mice.

Shintaro Yamamoto-Mizuma, et al. J Physiol. 2004 June 1;557(Pt 2):439-456.
3.
Figure 6

Figure 6. Comparison of the effects of PDBu and anti-ClC-3 (A1–14) Ab on native VSOACs in pulmonary artery smooth muscle cells (PASMCs) from Clcn3+/+ (WT) and Clcn3−/− mice (KO). From: Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice.

A, representative VSOAC currents recorded in isotonic, hypotonic and hypotonic +100 nm PDBu solutions. Membrane currents were elicited by 400 ms voltage steps ranging from −80 to +80 mV in 20 mV increments from a holding potential of −40 mV. B, mean current densities measured at −80 mV (downward bars) and +80 mV (upward bars) in isotonic, hypotonic, and hypotonic + PDBu solutions. C, mean current densities measured at +80 mV (upward bars) and −80 mV (downward bars) in isotonic and hypotonic solutions for PASMCs from WT and KO mice intracellularly dialysed with 1.5 μg ml−1 A1–14 Ab.

Shintaro Yamamoto-Mizuma, et al. J Physiol. 2004 June 1;557(Pt 2):439-456.
4.
Figure 3

Figure 3. PKC-activated CFTR currents in atrial myocytes from Clcn3−/− (KO) mice. From: Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice.

A, time course of changes of CFTR currents at +80 mV (•) and −80 mV (○) in atrial cells of KO mice before (arrow at time a), after exposure to 100 nm PDBu (arrow at time b) and after exposure to cAMP cocktail (0.5 mm 8-Br-cAMP +2.0 mm IBMX; arrow at time c). B, CFTR currents were activated by PDBu and further enhanced by cAMP cocktail. Currents were recorded at the time points (a, b and c) indicated in A; traces labelled ba and cb are PDBu- and cAMP cocktail-induced difference currents, respectively. C, mean IV relationships at the same time points (a, b and c) in A. PDBu activated CFTR current in 4 out of 15 cells, and cAMP cocktail enhancement was observed in all 4 cells. D, mean IV relationships of PDBu- (ba) and cAMP cocktail- (cb) induced difference currents.

Shintaro Yamamoto-Mizuma, et al. J Physiol. 2004 June 1;557(Pt 2):439-456.
5.
Figure 2

Figure 2. Comparison of endogenous PKC regulation of VSOAC currents in atrial myocytes from Clcn3+/+ (WT) and Clcn3−/− (KO) mice. From: Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice.

A and B, time course of inhibition of VSOAC currents at +80 mV (•) and −80 mV (○) in atrial cells of WT (A) and KO (B) mice exposed to hypotonic solution and the subsequent application of 100 nm PDBu. C and D, mean IV relationships of VSOAC currents from WT (n = 6) mice and KO (n = 7) mice at the time points (a and b) indicated in A and B. E and F, time course of activation of VSOAC currents at +80 mV (•) and −80 mV (○) in atrial myocytes of WT (E) and KO (F) mice in isotonic or hypotonic solution in the presence of 100 nm BIM. G and H, mean IV relationships of VSOAC currents from WT (n = 6) mice and KO (n = 7) mice, respectively, at the time points (a, b and c) indicated in E and F. I, mean IV relationships of BIM-induced VSOAC currents in myocytes from WT (•) mice and KO (○) mice in isotonic solution.

Shintaro Yamamoto-Mizuma, et al. J Physiol. 2004 June 1;557(Pt 2):439-456.
6.
Figure 5

Figure 5. Comparison of sensitivity to [ATP]i and free [Mg2+]i of VSOAC currents in atrial myocytes from Clcn3+/+ (WT) and Clcn3−/− (KO) mice. From: Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice.

A and B, time course of activation of VSOAC currents at +80 mV (•) and −80 mV (○) in atrial cells from WT and KO mice with ATP depletion (0 mm ATP in pipette solution (0 mm[ATP]i) and 0 mm glucose +2 mm NaCN in bathing solution). C and D, mean IV relationships of WT (n = 4) and KO (n = 4) cells at the time points (a, b and c) of indicated in A and B, respectively. E and F, time course of inhibition of VSOAC currents at +80 mV (•) and −80 mV (○) in myocytes from WT (E) and KO (F) with ATP depletion plus 2 mm intracellular free Mg2+ (high free [Mg2+]i). G and H, mean IV relationships of VSOAC currents from WT (n = 4) and KO (n = 4) mice at the time points indicated in E and F. I, mean IV relationships of hypotonic-activated difference currents in myocytes from WT (•) and KO (○) mice.

Shintaro Yamamoto-Mizuma, et al. J Physiol. 2004 June 1;557(Pt 2):439-456.
7.
Figure 8

Figure 8. Two-dimensional electrophoresis analysis of protein expression patterns in membranes of cardiac cells from Clcn3+/+ and Clcn3−/− mice. From: Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice.

A, representative 2-D gel depicts Coomassie-stained proteins from wild-type (Clcn3+/+) mouse heart. B, representative 2-D gel depicts Coomassie-stained proteins from Clcn3−/− mouse heart. C, spot sets created from images of 2-D gels of both wild-type and Clcn3−/− mouse heart run under the same conditions as the gels in A and B and compared using Bio-Rad PDQuest version 7.1.1 software. Three gels were run for each mouse heart type; two hearts were pooled to provide proteins for each gel. The filled symbols indicate changes in protein patterns in Clcn3−/− compared to wild-type. A total of 35 proteins consistently changed (minimum criteria: > 2-fold change) in membranes from Clcn3−/− mouse heart in all 3 experiments (6 missing proteins, 2 new proteins, 9 up-regulated proteins, 15 down-regulated proteins, and 2 translocated proteins). The open squares (□) in A, B, and C indicate the location (molecular mass 85 kDa and pI 6.9) of the ClC-3 protein spot (No. 3812) in the 2-D gels, which was independently confirmed by Western blotting using a specific anti-ClC-3 C670–687 antibody.

Shintaro Yamamoto-Mizuma, et al. J Physiol. 2004 June 1;557(Pt 2):439-456.
8.
Figure 1

Figure 1. Comparison of native VSOAC currents in cardiac atrial myocytes from Clcn3+/+ (WT) and Clcn3−/− (KO) mice. From: Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice.

A and B, time course of changes of VSOAC currents at +80 mV (•) and −80 mV (○) in atrial cells of WT (A) and KO (B) mice exposed to hypotonic (HYPO) and hypertonic (HYPER) solutions. The external solution initially was isotonic (ISO) solution. C and D, VSOAC currents in myocytes from WT mice (C) and from KO mice (D) in isotonic and hypotonic solutions, respectively. Currents were recorded at the time points (a and b) indicated in A and B. Here and in subsequent similar figures using atrial myocytes, the pulse protocol is the same as shown in the upper part of C; changes in whole-cell currents were monitored by applying 400 ms voltage-clamp steps to membrane potentials between −100 and +120 mV in +20 mV steps from a holding potential of −40 mV every 5 s. E, mean IV relationships of VSOAC currents from WT (n = 18) and KO (n = 6) myocytes in isotonic and hypotonic solutions. F and G, anion permeability of VSOACs from WT and KO myocytes. H, comparison of inhibition by Cl channel inhibitors (glibenclamide (0.2 mm), DIDS (0.1 mm), niflumic acid (0.1 mm) and extracellular ATP (1 mm)) of VSOACs in myocytes from WT and KO mice (n = 4 in each case); EH, values represent mean ± s.e.m.

Shintaro Yamamoto-Mizuma, et al. J Physiol. 2004 June 1;557(Pt 2):439-456.

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