High-affinity ouabain binding by a chimeric gastric H+,K+-ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the alpha 1-subunit of rat Na+,K+-ATPase

Abstract

Na(+),K(+)-ATPase and gastric H(+),K(+)-ATPase are two related enzymes that are responsible for active cation transport. Na(+), K(+)-ATPase activity is inhibited specifically by ouabain, whereas H(+),K(+)-ATPase is insensitive to this drug. Because it is not known which parts of the catalytic subunit of Na(+),K(+)-ATPase are responsible for ouabain binding, we prepared chimeras in which small parts of the alpha-subunit of H(+),K(+)-ATPase were replaced by their counterparts of the alpha(1)-subunit of rat Na(+),K(+)-ATPase. A chimeric enzyme in which transmembrane segments 5 and 6 of H(+), K(+)-ATPase were replaced by those of Na(+),K(+)-ATPase could form a phosphorylated intermediate, but hardly showed a K(+)-stimulated dephosphorylation reaction. When transmembrane segments 3 and 4 of Na(+),K(+)-ATPase were also included in this chimeric ATPase, K(+)-stimulated dephosphorylation became apparent. This suggests that there is a direct interaction between the hairpins M3-M4 and M5-M6. Remarkably, this chimeric enzyme, HN34/56, had obtained a high-affinity ouabain-binding site, whereas the rat Na(+), K(+)-ATPase, from which the hairpins originate, has a low affinity for ouabain. The low affinity of the rat Na(+),K(+)-ATPase previously had been attributed to the presence of two charged amino acids in the extracellular domain between M1 and M2. In the HN34/56 chimera, the M1/M2 loop, however, originates from H(+),K(+)-ATPase, which has two polar uncharged amino acids on this position. Placement of two charged amino acids in the M1/M2 loop of chimera HN34/56 results in a decreased ouabain affinity. This indicates that although the M1/M2 loop affects the ouabain affinity, binding occurs when the M3/M4 and M5/M6 hairpins of Na(+),K(+)-ATPase are present.

Figure 1

Schematic representation of the produced chimeras and wild-type H⁺,K⁺-ATPase. The open bars represent H⁺,K⁺-ATPase sequences, and the solid bars represent Na⁺,K⁺-ATPase sequences. HK, H⁺,K⁺-ATPase; NaK, Na⁺,K⁺-ATPase; HNN3, H⁺,K⁺-ATPase with amino acids Met1-Ile293 replaced by those of Na⁺,K⁺-ATPase (Met1-Ile279); HN36, H⁺,K⁺-ATPase with amino acids Ile293-Arg846 replaced by those of Na⁺,K⁺-ATPase (Ile279-Arg832); HN34, H⁺,K⁺-ATPase with amino acids Ile293-Leu346 replaced by those of Na⁺,K⁺-ATPase (Ile279-Leu332); HN56, H⁺,K⁺-ATPase with amino acids Leu776-Arg846 replaced by those of Na⁺,K⁺-ATPase (Leu762-Arg832); HN34/56, H⁺,K⁺-ATPase with amino acids Ile293-Leu346 and Leu776-Arg846 replaced by those of Na⁺,K⁺-ATPase (Ile279-Leu332 and Leu762-Arg832).

Figure 2

ATPase activity of the chimeras and wild-type H⁺,K⁺-ATPase. The assay was performed at 37°C with ≈1 μg of protein in the presence of 0.2 mM EDTA/0.1 mM EGTA/1.2 mM MgCl₂/50 mM Tris-acetic acid, pH 6.0/3.0 mM KCl/10 μM ATP. The ATPase activity determined was corrected for that of the mock. Shown are mean values ± SE of two to four enzyme preparations.

Figure 3

Effects of ouabain and vanadate on the ATPase activity of the chimeras and wild-type H⁺,K⁺-ATPase. The assay was performed as in Fig. 2, using varying concentrations of ouabain and vanadate. The ATPase activity determined was corrected for that of the mock. The H⁺,K⁺-ATPase activity in the absence of inhibitors was set at 100%. ■, H⁺,K⁺-ATPase; □, HNN3; ●, HN36; ○, HN34; ▴, HN56; ▿, HN34/56. Shown is the average of two enzyme preparations.

Figure 4

Phosphorylation capacity of the chimeras and wild-type H⁺,K⁺-ATPase. Sf9 membranes (≈5 μg) were preincubated at 21°C with 0.2 mM EDTA/1.2 mM MgCl₂/50 mM Tris-acetic acid, pH 6.0. After phosphorylation at 21°C for 10 s with 0.1 μM [γ-³²P]ATP, the phosphorylation level (EP) was determined and corrected for that of the mock. Shown are mean values ± SE of two to four enzyme preparations.

Figure 5

Effect of ouabain on the phosphorylation capacity of the chimeras and wild-type H⁺,K⁺-ATPase. Sf9 membranes were preincubated as in Fig. 4, using varying concentrations of ouabain. After phosphorylation at 21°C for 10 s with 0.1 μM [γ-³²P]ATP, the phosphorylation level (EP) was determined and corrected for that of the mock. The phosphorylation level of H⁺,K⁺-ATPase in the absence of ouabain was set at 100%. ■, H⁺,K⁺-ATPase; □, HNN3; ●, HN36; ○, HN34; ▴, HN56; ▿, HN34/56. Shown is the average of two enzyme preparations.

Figure 6

Effect of ouabain on the ATPase activity of the rat Na⁺,K⁺-ATPase, mutant Na⁺,K⁺-ATPase R113Q/D124N, chimera HN34/56, mutant HN34/56 Q127R, and mutant HN34/56 Q127R/N138D. The assay shown in A was performed in the presence of 0.2 mM EDTA/0.1 mM EGTA/1.2 mM MgCl₂/50 mM Tris-acetic acid, pH 7.0/10 mM KCl/100 mM NaCl/100 μM ATP. The assay conditions used for the assay shown in B were similar to those described in Fig. 2. The ATPase activity determined was corrected for that of the mock. The ATPase activity in the absence of ouabain was set at 100%: Na⁺,K⁺-ATPase, 0.37 μmol Pi mg⁻¹⋅protein h⁻¹ (■); NaKQN, 0.41 μmol Pi mg⁻¹⋅protein h⁻¹ (○); HN34/56, 0.10 μmol Pi mg⁻¹⋅protein h⁻¹ (○); HN34/56R, 0.08 μmol Pi mg⁻¹⋅protein h⁻¹ (▿); HN34/56RD, 0.04 μmol Pi mg⁻¹⋅protein h⁻¹ (■). Results are representative of two enzyme preparations.

Figure 7

[³H]Ouabain binding to the chimeras, mutants, and wild-type ATPases. Approximately 100 μg of protein was incubated at 21°C in the presence of 5.0 mM MgCl₂/50 mM Tris-acetic acid, pH 7.0/1.0 mM H₃PO₄ or 1.0 mM ATP and 10 nM [³H]ouabain. The determined level of bound ouabain was corrected for that of the mock. Shown are mean values ± SE of three enzyme preparations.

Figure 8

[³H]Ouabain binding to chimera HN34/56 and rat Na⁺,K⁺-ATPase R113Q/D124N. The enzymes were incubated at 21°C in the presence of 5.0 mM MgCl₂/50 mM Tris-acetic acid, pH 7.0/1.0 mM ATP/100 mM NaCl/10 nM [³H]ouabain and varying concentrations of nonradioactive ouabain. The binding of [³H]ouabain in the absence of nonradioactive ouabain was set at 100%: NaKQN, 0.19 ± 0.03 pmol [³H]ouabain mg⁻¹ protein (■); HN34/56, 0.14 ± 0.03 pmol [³H]ouabain mg⁻¹ protein (○). Results are the average of three enzyme preparations.