Does Binding of Ouabain to Human α1-Subunit of Na+,K+-ATPase Affect the ATPase Activity of Adjacent α1-Subunit?

Abstract

To ascertain whether ouabain binding to human alpha1-subunit influences coexpression of rat alpha1-subunit, the ouabain-sensitive profiles of Na+,K+-ATPase activity and 86Rb+ uptake activity and ouabain binding capacity were measured in HeLa cells stably expressing rat alpha1-subunit. The ouabain-sensitive profile of ATPase and 86Rb+ uptake activity seemed to be the sum of two components, one with high and one with low apparent affinity to ouabain, which were similar to that observed in HeLa and NRK-52E cells derived from human and rat, respectively. The ATPase activity with low sensitivity to ouabain increased in simple proportion to the amount of the rat alpha1 mRNA derived from transfected cDNA, which was determined by the reverse transcription-polymerase chain reaction method. The turnover number of the human Na+,K+-ATPase activity obtained from the ratio of the Na+,K+-ATPase activity to the ouabain binding capacity is about 150/sec. The expression of the rat alpha1-subunit had no effect on the turnover numbers of the Na+,K+-ATPase activity with high affinity to ouabain estimated from the ouabain binding capacity as the active site concentration. These results suggested that the ouabain bound to human alpha1-subunit did not inhibit the ATPase activity of the coexpressing rat alpha1 in these cells.

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

“…Site-directed Mutagenesis-Plasmids pGEM-NaK and pCDL-NaK containing the entire coding region of rat Na/K-ATPase ␣1 cDNA have been described previously (26). Site-directed mutagenesis was performed using the megaprimer PCR method (27) in conjunction with the following sense oligonucleotide primers: for G442A/P, 5Ј-GCA GTA GCG NCA GAT GCT TCC-3Ј; for S445A, 5Ј-GGA GAT GCT GCC GAG TCG-3Ј; for E446A, 5Ј-GCT TCC GCG TCG GCG CTC-3Ј and antisense primer for D443A, 5Ј-GGA AGC AGC TCC CGC TAC TGC-3Ј.…”

“…The mutations were verified by nucleotide sequencing (28). The entire coding region of the mutated rat ␣1 cDNA was transferred to the expression vector pCDL as described previously (26).…”

“…Preparation of SDS-purified Membrane Vesicles-Crude plasma membranes were prepared from HeLa cells (26) or pig kidney microsomes essentially as described previously (29). Crude plasma membranes at a concentration of 1 mg/ml were incubated with 0.15 mg/ml of SDS in a buffer containing 0.25 M sucrose, 10 mM dithiothreitol, and 10 mM Tris-HCl, pH 7.4, for 5 min at room temperature.…”

Replacement of Several Single Amino Acid Side Chains Exposed to the Inside of the ATP-binding Pocket Induces Different Extents of Affinity Change in the High and Low Affinity ATP-binding Sites of Rat Na/K-ATPase

“…Plasmids-pGEM-NaK and pCDL-NaK containing the entire coding region of rat Na ϩ /K ϩ -ATPase ␣1 cDNA has been described previously (24). The DNA fragments encoding the rat Na ϩ /K ϩ -ATPase ␤ subunit were amplified by the PCR using a pair of sense and antisense oligonucleotide primers: for sense, 5Ј-AGCACTCGCTTTCCCTC-3Ј, corresponding to the nucleotides 413-429; for antisense, 5Ј-GGTCCCAT-ACGTATGAC-3Ј, corresponding to the nucleotides 1455-1471 (25).…”

“…The mutations were verified by nucleotide sequencing. The entire coding region of the mutated rat ␣1 cDNA was transferred to the expression vector pCDL as described previously (24).…”