Nikolai N. Modyanov

Transport and Pharmacological Properties of Nine Different Human Na,K-ATPase Isozymes

Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3 α and β isoforms, were expressed in Xenopus oocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K+-activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the α isoform. On the other hand, variations in external K+ activation are determined by a cooperative interaction mechanism between α and β isoforms with α2-β2 complexes having the lowest apparent K+ affinity. α Isoforms influence the apparent internal Na+ affinity in the order α1 > α2 > α3 and the voltage dependence in the order α2 > α1 > α3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, α2-β isozymes exhibit more rapid ouabain association as well as dissociation rate constants than α1-β and α3-β isozymes. Finally, isoform-specific differences exist in the K+/ouabain antagonism which may protect α1 but not α2 or α3 from digitalis inhibition at physiological K+ levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.

Identification of a novel gene of the X,K-ATPase β-subunit family that is predominantly expressed in skeletal and heart muscles1

We have identified the fifth member of the mammalian X,K‐ATPase β‐subunit gene family. The human and rat genes are largely expressed in skeletal muscle and at a lower level in heart. The deduced human and rat proteins designated as βmuscle (βm) consist of 357 and 356 amino acid residues, respectively, and exhibit 89% identity. The sequence homology of βm proteins with known Na,K‐ and H,K‐ATPase β‐subunits are 30.5–39.4%. Unlike other β‐subunits, putative βm proteins have large N‐terminal cytoplasmic domains containing long Glu‐rich sequences. The data obtained indicate the existence of hitherto unknown X,K‐ATPase (most probably Na,K‐ATPase) isozymes in muscle cells.

Ouabain-sensitive H,K-ATPase: tissue-specific expression of the mammalian genes encoding the catalytic α subunit1

Human ATP1AL1 and corresponding genes of other mammals encode the catalytic α subunit of a non‐gastric ouabain‐sensitive H,K‐ATPases, the ion pump presumably involved in maintenance of potassium homeostasis. The tissue specificity of the expression of these genes in different species has not been analyzed in detail. Here we report comparative RT‐PCR screening of mouse, rat, rabbit, human, and dog tissues. Significant expression levels were observed in the skin, kidney and distal colon of all species (with the exception of the human colon). Analysis of rat urogenital organs also revealed strong expression in coagulating and preputial glands. Relatively lower expression levels were detected in many other tissues including brain, placenta and lung. In rabbit brain the expression was found to be specific to choroid plexus and cortex. Prominent similarity of tissue‐specific expression patterns indicates that animal and human non‐gastric H,K‐ATPases are indeed products of homologous genes. This is also consistent with the high sequence similarity of non‐gastric H,K‐ATPases (including partial sequences of hitherto unknown cDNAs for mouse and dog proteins).

Intersubunit and intrasubunit contact regions of Na+/K(+)-ATPase revealed by controlled proteolysis and chemical cross-linking.

To identify interfaces of α- and β-subunits of Na+/K+-ATPase, and contact points between different regions of the same α-subunit, purified kidney enzyme preparations whose α-subunits were subjected to controlled proteolysis in different ways were solubilized with digitonin to disrupt intersubunit α,α-interactions, and oxidatively cross-linked. The following disulfide cross-linked products were identified by gel electrophoresis, staining with specific antibodies, and N-terminal analysis. 1) In the enzyme that was partially cleaved at Arg438-Ala439, the cross-linked products were an α,β-dimer, a dimer of N-terminal and C-terminal α fragments, and a trimer of β and the two α fragments. 2) From an extensively digested enzyme that contained the 22-kDa C-terminal and several smaller fragments of α, two cross-linked products were obtained. One was a dimer of the 22-kDa C-terminal peptide and an 11-kDa N-terminal peptide containing the first two intramembrane helices of α (H1-H2). The other was a trimer of β, the 11-kDa, and the 22-kDa peptides. 3) The cross-linked products of a preparation partially cleaved at Leu266-Ala267 were an α,β-dimer and a dimer of β and the 83-kDa C-terminal fragment. Assuming the most likely 10-span model of α, these findings indicate that (a) the single intramembrane helix of β is in contact with portions of H8-H10 intramembrane helices of α; and (b) there is close contact between N-terminal H1-H2 and C-terminal H8-H10 segments of α; with the most probable interacting helices being the H1,H10-pair and the H2,H8-pair.

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na+, K+‐ATPase

Purified kidney Na+,K+‐ATPase whose α‐subunit is cleaved by chymotrypsin at Leu266‐Ala267, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of α‐cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the α‐subunits of oligomeric membrane‐bound enzyme are dormant and that interaction of the N‐terminal domain of α‐subunit with its phosphorylation domain causes this half‐site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all α‐subunits.

Ligand-sensitive Interactions among the Transmembrane Helices of Na+/K+-ATPase

An extensively trypsin-digested Na+/K+-ATPase, which retains the ability to bind Na+, K+, and ouabain, consists of four fragments of the α-subunit that contain all 10 transmembrane α domains, and the β-subunit, a fraction of which is cleaved at Arg142-Gly143. In previous studies, we solubilized this preparation with a detergent and mapped the relative positions of several transmembrane helices of the subunits by chemical cross-linking. To determine if these detected helix-helix proximities were representative of those existing in the bilayer prior to solubilization, we have now done similar studies on the membrane-bound preparation of the same digested enzyme. After oxidative sulfhydryl cross-linking catalyzed by Cu2+-phenanthroline, two prominent products were identified by their mobilities and the analyses of their N termini. One was a dimer of a 11-kDa α-fragment containing the H1-H2 helices and a 22-kDa α-fragment containing the H7-H10 helices. This dimer seemed to be the same as that obtained in the solubilized preparation. The other product was a trimer of the above two α-fragments and that fraction of β whose extracellular domain was cleaved at Arg142-Gly143. This product was different from a similar one of the solubilized preparation in that the latter contained the predominant fraction of β without the extracellular cleavage. The cross-linking reactions of the membrane preparation, but not those of the solubilized one, were hindered specifically by Na+, K+, and ouabain. These findings indicate that (a) the H1-H2 transmembrane helices of α are adjacent to some of its H7-H10 helices both in solubilized and membrane-bound states, (b) the alignment of the residues of the single transmembrane helix of β with the interacting H1-H2 and H7-H10 helices of α is altered by detergent solubilization and by structural changes in the extracellular domain of β, and (c) the three-dimensional packing of the interacting transmembrane helices of α and β are regulated by the specific ligands of the enzyme.

Nongastric H,K-ATPase: structure and functional properties.

Abstract: Nongastric H,K‐ATPases whose catalytic subunits (AL1) encoded by human ATP1AL1 and homologous animal genes comprise the third distinct group within the X,K‐ATPase family. No unique nongastric β has been identified. Precise in situ colocalization and strong association of AL1 with β1 of Na,K‐ATPase was detected in apical membranes of rodent prostate epithelium. In this tissue, β1NK serves as an authentic subunit of both the Na,K‐ and nongastric H,K‐pumps. Upon expression in Xenopus oocytes the human AL1 can assemble with β1NK, and more efficiently with gastric βHK, into functional H,K‐pumps. Both AL1/β complexes exhibit a similar K‐affinity, and their K‐transport depends on intra‐ and extracellular Na. These data provide new evidence that nongastric H,K‐ATPase can perform Na/K‐exchange, and indicate that β does not significantly affect this ion‐pump function. Analysis of human nongastric H,K‐ATPase expressed in Sf‐21 insect cells revealed that AL1/βHK exhibits substantial enzymatic activities in K‐free medium and K stimulates, but Na has inhibitory effect on ATP hydrolysis. Thus, although the nongastric H,K‐ATPase can function as Na/K exchanger, its reaction mechanism is different from that of the Na,K‐ATPase. Human nongastric H,K‐ATPase is highly sensitive to bufalin, digoxin, and digitoxin, but almost resistant to digoxigenin and ouabagenin.

Tissue Specificity of Alternative Splicing of Transcripts Encoding Hampin, a New Mouse Protein Homologous to the Drosophila MSL-1 Protein

A number of mammalian genomes have one gene copy encoding the protein that we named hampin. A search in a number of databases revealed a distant homologue, the well-known Drosophila protein MSL-1 (male-specific lethal 1). An alternative splicing of mRNA led to a significant diversity of structural hampin variants with different domain compositions. We analyzed the tissue-specific expression of five mouse hampin variants using RT-PCR. Two variants encoding hampin proteins with truncated N termini were shown to have a restricted tissue specificity: they are exclusively expressed in the testes. The mRNAs of other hampin variants were detected in all the tested tissues at comparable levels. We obtained polyclonal antibodies to the recombinant hampin and used them to demonstrate that at least one of the variants is predominantly localized in the nucleus. The specific features of the hampin primary structure and its possible functions as a member of the hampin/MSL-1 family of proteins are discussed.

Human Nongastric H,K-ATPase: Current View On Structure And Functional Properties

The P-type ATPases comprise a large number of highly diverse transport ATPases that are predominantly involved in the active transport of cations across biological membranes. All of these ion pumps share a common feature: formation of a phosphorylated intermediate during the reaction cycle (26,39). Different K+-dependent animal ATPases (X,K-ATPases) are the most closely related among various P-type ATPases. All of the known X,K-ATPases function as cation exchangers that pump K+ into the cell and Na+ or H+ out of the cell. X,K-ATPases exhibit a much higher level of sequence homology between their catalytic α-subunits than with other P-ATPases, and contain a second component, a s-subunit which is absent in other P-ATPases (26,39,53). The catalytic α-subunits are large polytopic proteins (~110 kDa) with 10 transmembrane segments and contain most of the ATPase functional domains such as the ATP-hydrolyzing center and the binding sites for cations and specific inhibitors (8,26,53). The glycosylated s-subunits (core protein ~ 30–35 kDa) have a relatively short cytoplasmic N-terminal domain, a single transmembrane segment, and a large ectodomain containing three conserved disulfide bridges and several carbohydrate chains (11,19). The X,K-ATPase family combines three distinct groups of ion pumps. Two groups, one consisting of the Na,K-ATPase isozymes formed by four α three s isoforms and the second which includes the gastric H,K-ATPase, have long been known and studied extensively (26,53). The recently discovered catalytic a-subunits of nongastric H,K-ATPases encoded by the human ATP1 AL1 (alternative name ATP12A) gene and its animal homologues represent the third distinct group (23,27,36).

Structural analysis of the products of chymotryptic cleavage of the E1 form of Na,K-ATPase α-subunit: identification of the N-terminal fragments containing the transmembrane H1-H2 domain

Chymotryptic cleavage of the Na,K‐ATPase in NaCl medium abolishes ATPase activity and alters other functional parameters. The structure of this modified enzyme is uncertain since only one product of selective proteolysis, the 83‐kDa fragment of the α‐subunit (Ala267–C‐terminus) has been identified previously. Here, we applied additional tryptic digestion followed by oxidative cross‐linking to identify the products originating from the N‐terminal part of the α‐subunit. These fragments start at Ala72 or Thr74 and contain the transmembrane H1‐H2 domain. Formation of cross‐linked product between α‐fragments containing H1‐H2 and H7‐H10 demonstrate that the structural integrity of the membrane moiety is preserved. We also determined that secondary cleavage of the 83‐kDa fragment leads to the formation of C‐terminal 48‐kDa α‐fragments with multiple N‐termini at Ile582, Ser583, Met584 and Ile585.