Chemical modification of Glu-953 of the alpha chain of Na+,K(+)-ATPase associated with inactivation of cation occlusion.

Abstract

We have investigated the role, number, and identity of glutamate (or aspartate) residues involved in cation occlusion on Na+,K+-ATPase, using the carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD). Extensive use is made of selectively trypsinized Na+,K+-ATPase--the so-called "19-kDa membranes"-containing a 19-kDa COOHterminal, smaller (8-11 kDa) membrane-embedded fragments of the a chain, and a largely intact ( measured by examination of labeling of 19-kDa peptide purified from "19-kDa membranes" or of a chain purified from native enzyme. Stoichiometries, estimated by extrapolation, are as follows: (for "19-kDa membranes") close to one DCCD per Rb+ site and one DCCD per 19-kDa peptide; and (for native enzyme) close to two DCCD per phosphoenzyme and two DCCD per a chain. We suggest that each of two K+ (or Na+) sites contains a carboxyl group, one located in the 19-kDa peptide and one elsewhere in the a chain. After cyanogen bromide digestion of purified, labeled a chain, or of 19-kDa peptide, a labeled fragment of apparent Mr -4 kDa was detected and was identified as that with NH2-terminal Lys-943.Rb+-protected []4CJDCCD incorporation was associated almost exclusively with Glu-953. We suggest that the cation occlusion "cage"l consists of ligating groups donated by different trans-membrane segments and includes two carboxyl groups such as Glu-953 (and perhaps Glu-327) as well as neutral groups, in two K+ (or Na+) sites, but only neutral groups in the third Na+ site.Understanding energy transduction by cation pumps requires knowledge of structure of the cation and ATP sites and their interactions. The structure of the cation sites is largely unknown. By analogy with well-characterized cation-binding proteins, bound or occluded cations are likely to be largely dehydrated and ligated with six to eight oxygens of carboxyls and neutral groups. Some information on cation-binding residues in pumps comes from chemical modification, sitedirected mutagenesis, and charge-transfer studies (for review, see ref.

A Glu329→ Gln variant of the α‐subunit of the rat kidney Na+K+‐ATPase can sustain active transport of Na+ and K+ and Na+K+‐activated ATP hydrolysis with normal turnover number

“…While the present study shows that Na' and K' binding take place in the Glu329+Gln variant, it does not exclude that the residue modified with DCCD in [5] was G~u~*~. The bulky DCCD molecule might have disturbed ion binding and occlusion, even if the modified residue were located at the periphery of the cation-binding pocket without being an essential component.…”

“…Moreover, it was found that each mol of asubunit bound 2 mol of DCCD. One labeled residue was located in a cyanogen bromide fragment of apparent M, 4 kDa, and [r4C]DCCD incorporation was found to be associated almost exclusively with G1u953 [5]. The role of G1u953 was, however, tested in a recent mutagenesis study, and it was shown that cation stimulation of Na',K+-ATPase activity was unaffected by single amino acid substitutions of G1u953 [16].…”

“…Further examination of the library revealed, in addition to this wild-type cDNA, a variant Na',K'-ATPase cDNA containing a single G-to-C base substitution, which on amino acid level gave rise to a glutamate-to-glutamine substitution at position 329 within the motif 32%PEGL in the predicted 4th transmembrane helix. As G1u3*' has recently been attributed a role in cation binding [5], it was pertinent to examine whether the Glu32g+Gln variant was able to carry out active transport of Na' and K' and Na+,K+-activated ATP hydrolysis. The cDNAs encoding either the Glu32g+Gln variant or the wild-type rodent Na',K'-ATPase were therefore inserted in the expression vector pMT2 and transfected into COS-1 cells, which were grown in the presence of 5 PM ouabain.…”

“…To identify within the tryptic fragments of Na+,K+-ATPase the residues essential for cation occlusion, Karlish and coworkers combined selective tryptic digestion with chemical modification using the hydrophobic carbodiimide DCCD [5,15]. It was observed that Rb' and Na' occlusion was inactivated by DCCD with identical rates, and the inactivation was equally well protected by Rb' with high affinity and Na' with lower affinity.…”

“…The role of G1u953 was, however, tested in a recent mutagenesis study, and it was shown that cation stimulation of Na',K+-ATPase activity was unaffected by single amino acid substitutions of G1u953 [16]. It was not possible to determine the exact location of the second DCCD binding site, but peptides corresponding to the transmembrane hairpin loop Mr plus M, or M, plus M, appeared to be labeled, and GIu~*~ was suggested as the residue most likely labeled by DCCD [5]. This was based on assumption that DCCD labeling occurs at a carboxyl group and on extrapolation to the Na+,K+-ATPase of the mutagenesis data obtained with the homologous residue in the Ca*'-ATPase.…”