The Na-K-ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both its α- and β-subunits. At present, a...

Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function

The energy dependent exchange of cytoplasmic Na+ for extracellular K+ in mammalian cells is due to a membrane bound enzyme system, the Na,K-ATPase. The exchange sustains a gradient for Na+ into and for K+ out of the cell, and this is used as an energy source for creation of the membrane potential, for its de- and repolarisation, for regulation of cytoplasmic ionic composition and for transepithelial transport. The Na,K-ATPase consists of two membrane spanning polypeptides, an α-subunit of 112-kD and a β-subunit, which is a glycoprotein of 35-kD. The catalytic properties are associated with the α-subunit, which has the binding domain for ATP and the cations. In the review, attention will be given to the biochemical characterization of the reaction mechanism underlying the coupling between hydrolysis of the substate ATP and transport of Na+ and K+.

The Na,K-ATPase.

The primary active transport of Na +, K +, Ca 2T and H + in eukaryotic cells is driven by ATP-powered cation pumps, the Na,K pump, Ca pump and H,K pump. The intermediary steps of the pump reactions and their relationship to cation translocation have been examined in detail, particularly for the Na,K-pump and Ca-pump proteins. A common feature of these pumps is a protein of Mr close to 110,000 that binds ATP and accepts its y-phosphate in a covalent Asp-P bond. There is evidence that both phosphoand dephospho-forms of the protein exist in two major conformational states, E1 and E2, with different affinities and orientation of cation binding sites, and ATP-driven cation pumping is blocked by micromolar concentrations of vanadate [39, 68, 95, 96, 116, 132]. A wealth of structural information about the cation pump proteins appeared recently after identification of mRNA and sequencing of cloned cDNA. Several isoforms of the genes of o~-subunit of Na,K pump were identified in the human and rat genomes [147, 182] and the sequences ofa-subunit [110, 111,145, I46, 178,181] and B-subunit [22, 112, 140, 146, 179] were deduced from cDNA of mRNA from a variety of tissues in piscine and mammalian species. A similar intensity of work in adjacent fields provided the sequences of slow and fast twitch Ca pump from sarcoplasmic reticulum [19, 121] and H,K pump from stomach mucosa [180]. In addition the gene sequences are available for K pumps [82, 185] and H pumps [1, 177] from microorganisms. As paradigm for interpretation of this information the combined X-ray crystallographic structure at high resolution and

Structural basis for E1-E2 conformational transitions in Na,K-pump and Ca-pump proteins.

THE (Na+ + K+)ATPase, an integral membrane protein located in virtually all animal cells, couples the hydrolysis of ATP to the countertransport of Na+ and K+ ions across the plasma membrane1,2. In neurons, a large portion of cellular energy is expended by this enzyme to maintain the ionic gradients that underlie resting and action potentials. Although neurotransmitter regulation of the enzyme in brain has been reported3,4, such regulation has been characterized either as a nonspecific phenomenon5,6 or as an indirect effect of neurotransmitter-induced changes in ionic gradients7. We report here that the neurotransmitter dopamine, through a synergistic effect on D1 and D2 receptors, inhibits the (Na+ + K+)ATPase activity of isolated striatal neurons. Our data provide unequivocal evidence for regulation by a neurotransmitter of a neuronal ion pump. They also demonstrate that synergism between D1 and D2 receptors, which underlies many of the electrophysical and behavioural effects of dopamine in the mammalian brain8, can occur on the same neuron. In addition, the results support the possibility that dopamine and other neurotransmitters can regulate neuronal excitability through the novel mechanism of pump inhibition.

Inhibition by dopamine of (Na(+)+K+)ATPase activity in neostriatal neurons through D1 and D2 dopamine receptor synergism.

P1B-type ATPases transport a variety of metals (Cd2+, Zn2+, Pb2+, Co2+, Cu2+, Ag+, Cu+) across biomembranes. Characteristic sequences CP[C/H/S] in transmembrane fragment H6 were observed in the putative transporting metal site of the founding members of this subfamily (initially named CPx-ATPases). In spite of their importance for metal homeostasis and biotolerance, their mechanisms of ion selectivity are not understood. Studies of better-characterized P(II)-type ATPases (Ca-ATPase and Na,K-ATPase) have identified three transmembrane segments that participate in ion binding and transport. Testing the hypothesis that metal specificity is determined by conserved amino acids located in the equivalent transmembrane segments of P1B-type ATPases (H6, H7, and H8), 234 P1B-ATPase protein sequences were analyzed. This showed that although H6 contains characteristic CPX or XPC sequences, conserved amino acids in H7 and H8 provide signature sequences that predict the metal selectivity in each of five P1B-ATPase subgroups identified. These invariant amino acids contain diverse side chains (thiol, hydroxyl, carbonyl, amide, imidazolium) that can participate in transient metal coordination during transport and consequently determine the particular metal selectivity of each enzyme. Each subgroup shares additional structural characteristics such as the presence (or absence) of particular amino-terminal metal-binding domains and the number of putative transmembrane segments. These differences suggest unique functional characteristics for each subgroup in addition to their particular metal specificity.

Identification of Ion-Selectivity Determinants in Heavy-Metal Transport P1B-type ATPases

The Na+,K+-transporting adenosine triphosphatase (Na+,K+-ATPase), also often known as the sodium pump or sodium-potassium pump, is an enzyme, found in nearly all animal-cell membranes, that uses energy from the hydrolysis of intracellular ATP to transport Na+ ions outwards and K+ ions inwards. It may be thought of as having three substrates (ATP, intracellular Na+ ions, and extracellular K+ ions) and four products (ADP, orthophosphate, extracellular Na+ ions, and intracellular K+ ions.) Because more Na+ ions are pumped out than K+ ions are pumped in, the activity of the enzyme generates an outward movement of positive charge, and this outward current may also be considered a “product” of the reaction.

I. M. Glynn

Papers

The Na+, K+-Transporting Adenosine Triphosphatase