CPZ decreases K⁺-dependent Na⁺, K⁺-ATPase transport activity.

<p><b>A.</b> Raw trace of the effects of 30 µM of CPZ on Na⁺, K⁺-ATPase I_p in adult rat cardiac myocytes. The horizontal bold lines indicate the addition and wash-off of 5 mM KCl or the addition of CPZ, as indicated. <b>B.</b> Steady state current obtained following 5 and 10 min incubation with CPZ, compared to controls not treated with CPZ. <b>C.</b> Changes in K⁺-independent I_p following the addition of CPZ. <b>D.</b> The CPZ-induced changes of Na⁺, K⁺-ATPase I_p in adult rat cardiac myocytes are reversible. The data represent cells isolated from at least 6 individual animals, (number of cells shown inside bars; 1 min time point not shown) and are expressed as mean ±SEM (*significantly different from control, <i>P</i><0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test in panel B and by unpaired <i>t</i>-test in panel C.</p

Effect of CPZ on <i>p</i>NPP hydrolysis by Na⁺,K⁺-ATPase.

<p><i>p</i>NPPase activity was measured in the presence of histidine buffer (with the pH adjusted with Tris/HCl), 10 mM MgCl₂, 10 mM Na₂⁺-<i>p</i>NPP, 100 mM NaCl (A) or 100 mM KCl (B) in the presence of the indicated CPZ concentrations and in the absence of ATP. Released <i>p</i>NP was measured in a spectrophotometer at 410 nm as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096909#s2" target="_blank">Materials and Methods</a>. Data are expressed as µmol hydrolyzed <i>p</i>NPP · h⁻¹ · mg⁻¹ (at 24°C). The black circles depict the effect of linoleic acid on <i>p</i>NPPase activity in the presence of 100 mM Na⁺ at concentrations identical to those of CPZ. Note that the residual activity seen in panel B at high CPZ concentrations is likely due to the presence of 20 mM Na⁺ added with <i>p</i>NPP.</p

Proteolytic cleavage site in the E₁ structure of the Na⁺,K⁺-ATPase.

<p>Architecture of the cytoplasmic loop between membrane spans 6 and 7, as well as the cytoplasmic part of membrane spans 8–10 together with the C-terminal extension in the E₁·AlF₄⁻·ADP·3Na⁺ (PDB accession nr 3WGU). The figure was made using Pymol (<a href="http://www.pymol.com" target="_blank">www.pymol.com</a>). D926, which coordinates Na⁺ in the unique site, is shown in red. Part of the intracellular loop between membrane spans 6 and 7 (L67) is shown. This part contains an asparagine residue (N831, shown in white), cleavage at which produces the C-terminal 19 kDa fragment of the α-subunit [Ref. 38]. Positive amino acids in the C-terminal part of the α-subunit are shown in blue. T₄ indicates the cleavage site described in this study, occurring between R1005 and P1006, as evidenced from Edman degradation. Part of M9, shown in cyan, appears behind M8. The dashed double-headed arrow indicates hypothetical movements of M10 that would result in deflection of the C-terminal tail and consequently modification of the ion binding sites.</p

Effect of CPZ on oxonol VI fluorescence in the presence of 10 folds ion gradient across liposomal membrane.

<p>Liposomes were equilibrated with either 300(A) or 300 mM NaCl (B). The medium contained 30 mM histidine, 2 mM MgCl₂ and 20 µl proteoliposomes. CPZ was added first, followed by 75 µl Tris-ATP, as indicated by the arrows.</p

Trypsin cleavage in the presence of substrate ions.

<p>Trypsin cleavage of pig kidney Na⁺,K⁺-ATPase was performed in the presence of Tris buffer and 50 mM of NaCl, Gua⁺ hydrochloride, or KCl. The enzyme (100 µg) was allowed to equilibrate with ions for 30 min at 20°C, following by the addition of trypsin (trypsin/protein ratio ∼1∶100). In controls (Cnt), water replaced trypsin. The reaction was allowed to proceed for 40 min and was terminated using SDS sample buffer acidified with TCA. Protein fragments were resolved using SDS-PAGE and visualized by Western blotting using anti KETYY antibody. Each reaction was performed in the presence of Tris buffer with the indicated pH values. Representative of four different experiments is shown. The right labels indicate the symbols of cleavage sites introduced by Jørgensen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096909#pone.0096909-Jrgensen1" target="_blank">[29]</a>, together with the new T₄ site. Note that the very faint bands appearing at 4 kDa in the presence of Na⁺ or K⁺ likely represent conformational fluctuations of the protein during incubation with the protease (Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096909#pone.0096909-Inesi1" target="_blank">[58]</a>).</p

CPZ increases K⁺-independent Na⁺, K⁺-ATPase activity.

<p><b>A</b>. Raw trace of the effects of treatment with 30 µM of CPZ on K⁺-independent I_p followed by the addition of 10 mM ouabain. <b>B</b>. Changes in steady state K⁺-independent I_p as a result of CPZ and CPZ plus ouabain, normalized to the K⁺-free control I_p. The data represent cells isolated from 6 individual animals [number of cells are shown inside bars] and are expressed as mean ±SEM (*significantly difference from control, <i>P</i><0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test.</p

Effect of CPZ on oxonol VI fluorescence under Na⁺,K⁺ exchange conditions.

<p>Liposomes containing 30₂ were used and the experiment was performed as in A. Extravesicular K⁺ was diluted more than 70 folds following addition of liposomes to the medium. The ATP concentration used to start the reaction was 75 µM. The numbers indicate the percentage decrease in maximum fluorescence occurred by pretreatment with 25 µM ADP. P₁ and P₂ denote the rapid and the slow phases of fluorescence response occurring in the presence of extracellular (intravesicular) K⁺.</p

Effect of CPZ on cleavage of the C-terminal tail.

<p>Trypsin treatment, SDS-PAGE, and immunoblotting were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096909#pone-0096909-g007" target="_blank">Fig. 7</a>. The effect of CPZ on accumulation of the C-terminal fragment was investigated at pH 8 in the presence of either Na⁺ or Gua⁺. Note the significant difference between Na⁺ and Gua⁺ on the accumulation of the 55 kDa fragment, occurring at the middle of the α-subunit (T₁ site, Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096909#pone.0096909-Jrgensen1" target="_blank">[29]</a>). The anomalous migration of the 4 kDa peptide is possibly due to its polar properties and its small molecular mass. Note that, due to the difference in staining intensity, the upper part of the immunoblot was made less intense.</p

Effect of temperature, pH, and vanadate sensitivity of the CPZ treated enzyme.

<p><b>A.</b> Pig kidney Na⁺,K⁺-ATPase activity was measured at four different temperatures, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096909#s2" target="_blank">Materials and Methods</a>, in the presence of the indicated CPZ concentrations. The reaction contained 30 mM histidine buffer, pH 7.2, 100 mM NaCl, 20 mM KCl, 3 mM MgCl₂, and 3 mM ATP. The inhibition curves were analyzed using the Hill equation, giving the following IC₅₀ values; squares, 5°C (7.64±1.14 µM); circles, 15°C (13.88±1.05 µM); diamonds, 30°C (37.33±1.08 µM); and triangles, 37 °C (108.20±1.31 µM). <b>B.</b> Effect of pH on CPZ inhibition of Na⁺-ATPase activity. Na⁺-ATPase activity was measured as described in panel A, but in the absence of K⁺ and in the presence of different pH values (the reaction was buffered with 30 mM Tris adjusted with HCl), in the presence of the indicated CPZ concentrations. Squares, circles, and diamonds indicate measurements performed at pH 6, 7, or 8, respectively. <b>C</b>. Na⁺,K⁺-ATPase (NKA) activity was measured as in panel A, but in the presence of 1 mM ATP and the indicated vanadate concentrations at 23°C. The ATPase mixture contained either DMSO (squares) or 20 µM CPZ (circles).</p

Effect of CPZ on ASG II fluorescence in cultured HEK293 cells.

<p>Cultured cells were suspended in Tyrode buffer and loaded with ASG II prior to fluorescence measurements, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096909#s2" target="_blank">Materials and Methods</a>. Fluorescence was measured at 535 nm in the presence of 4 mM K⁺ (A), or in the absence of K⁺ (B) on the extracellular side. Extracellular Na⁺ was 140 mM in both cases. Fluorescence, indicative of changes in intracellular Na⁺ concentration, was measured as a function of time, following treatment of cells with DMSO (black squares), 10 µM CPZ (blue squares), or 30 µM CPZ (red squares).</p