Involvement of the M7/M8 extracellular loop of the sodium pump alpha subunit in ion transport. Structural and functional homology to P-loops of ion channels.
Mutations were introduced in the motif 884DDRW887 from an extracellular peptide of the sodium pump alpha subunit localized between M7 and M8 membrane spans to investigate a possible role of this structure in ion recognition. A homologous sequence 399QDCW402 that occurs in the P-loops of Na+ channels was shown earlier to be important for ion gating. Mutant sodium pumps were expressed in yeast and subsequently investigated for their behavior toward ouabain, Na+, K+, and ATP. Native enzyme and D884A, D884R, D885A, D885E, or D885R mutants all bind ouabain in the presence of phosphate and Mg2+. The KD values determined from Scatchard analysis are in the range 5-8 nM for the native enzyme and the D884A, D885E, or D885A mutants, and 15.7 +/- 2.04 and 30.1 +/- 4.32 nM for mutants D884R and D885R, respectively. This ouabain binding is reduced in the presence of K+ in a similar way for both native or mutant sodium pumps with relative affinities (K0.5) for K+ ranging from 1.4 to 3.7 mM. Ouabain binding in the presence of 100 microM ATP is promoted by Na+ with K0.5 = 1.64 +/- 0.01 mM for the native enzyme and K0.5 = 8. 6 +/- 1.35 mM for the D884R mutant. The K0.5 values of the two enzymes for ATP are 0.66 +/- 0.16 microM and 1.1 +/- 0.12 microM, respectively. Ouabain binding as a function of Na+ concentration, on the other hand, is very low for the D885R mutant, even at an ATP concentration of 2 mM. Phosphate or eosin, however, are recognized by this mutant enzyme, so that a major conformational change within the ATP-binding site appears unlikely. The inability of the D885R mutant to bind ouabain in the presence of Na+ and ATP could be explained by assuming that the M7/M8 connecting extracellular loop, which also contains the mutated amino acids, is invaginated within the plane of the plasma membrane and possibly involved in acceptance and/or release of Na+ ions coming from cytosolic areas of the protein. In this case, the placement of an additional positive charge might repel Na+ ions and interrupt their flow, thus not allowing the enzyme to assume the proper conformational state for ouabain binding. Such invaginated hydrophilic protein structures, such as the P-loops of Na+ and K+ channels, are already known and have been shown to participate in ion conduction.
Schneider
H
H
Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany.
Scheiner-Bobis
G
G
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D004801:Eosine Yellowish-(YS) / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry*; D017136:Ion Transport; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010042:Ouabain / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship
D007473:Ion Channels; D010710:Phosphates; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium; D004801:Eosine Yellowish-(YS)