Critical effects on catalytic function produced by amino acid substitutions at Asp⁸⁰⁴ and Asp⁸⁰⁸ of the al isoform of Na,K‐ATPase

Abstract

At two intramembrane carboxyl-containing amino acids of the sheep al isoform of Na,K-ATPase (Aspso and Aspass), both charge-conserving (Asp to Glu) and charge-deleting (Asp to Asn, Leu and Ala) replacements were made and the altered enzymes studied. Nucleotide changes encoding the amino acid substitutions were placed in a cDNA encoding a ouabainresistant enzyme (sheep al RD) and the encoded enzymes were expressed in ouabain-sensitive HeLa cells. Transfections with cDNAs carrying all As so4 L carrying AspsesAla, Asp substitutions, along with those Asn, and AspSOSLeu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Aspse4 and AspSo*. Only the expression of the Aspso8Glu enzyme produced ouabain-resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Aspsa4Ala and AspsasAla were expressed using an alternative selection system (the protein carrying the amino acid substitution was the ouabain-sensitive wild-type sheep al Na,K-ATPase, which was expressed in ouabain-resistant 3T3 cells), intact cells were able to bind extracellular ouabain with high affinity (& = l-30 nM), indicating that the inactive proteins were synthesized and folded properly in the plasma membrane. The results demonstrate that carboxyl side chains at positions 8oJ and 808 are critical for enzyme catalytic function.

Asp804 and Asp808 in the Transmembrane Domain of the Na,K-ATPase α Subunit Are Cation Coordinating Residues

“…It has previously been shown that replacement of Asp 804 with Ala, Leu, Asn, or Glu, and Asp 808 with Ala, Leu, or Asn results in proteins that are unable to support cell growth when expressed in HeLa or COS cells (18,21,25,27). Thus, these substituted proteins could not be characterized in these expression systems.…”

“…In this system ouabain binding at nanomolar concentrations of [ 3 H]ouabain was used to probe the exogenous protein without interference from the endogenous mouse Na,KATPase. Ouabain binding is an indication of the enzyme's overall structural integrity and under appropriate conditions provides information on the enzyme localization (20,27). Furthermore, the kinetics of ouabain binding depend on the different conformational states that the Na,K-ATPase can assume (38 -41).…”

“…All substitutions introduced in these positions, except for the conservative replacement, Asp 808 3 Glu, have produced nonfunctional enzymes, i.e. enzymes unable to support cell growth (18,21,(25)(26)(27). Although these studies have indicated that these two residues may be essential for enzyme function, the structural-functional role of the carboxyl side chains could not be discerned.…”

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

“…Studies on the former enzyme have shown that the carboxyl-selective reagent 4-(diazomethyl)-7-(diethylamino)-coumarin disrupts K ϩ and Na transport (30,31,33,43). Taken together, these findings imply that the M5 glutamyl residue is directly involved in transport by the mammalian Na -ATPase, both of which are required for high affinity cation binding and transport (30,31,33,39,41,43). Unlike its homologues, however, Asp-730 of the H ϩ -ATPase is not detectably involved in transport, since it can be replaced by Ala (as part of an R695A/ D730A double mutant) with little or no effect on ATP hydrolysis or ATP-dependent proton pumping.…”

Abolishment of Proton Pumping and Accumulation in the E1P Conformational State of a Plant Plasma Membrane H+-ATPase by Substitution of a Conserved Aspartyl Residue in Transmembrane Segment 6

“…For example, most of the extramembraneous regions of the Na ϩ /K ϩ -ATPase can be removed by proteolytic digestion while the remaining portion of the enzyme maintains its ability to bind and occlude K ϩ (10). Furthermore, mutagenesis studies have identified a number of amino acid residues within M4, M5, and M6 of the Na ϩ /K ϩ -ATPase that appear to be critical for cation binding (11)(12)(13)(14). These residues are in general polar, and most are negatively charged.…”