Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoforms α1, α2, α3 and the ATP1A3 promoter.

Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

The rapid-onset dystonia parkinsonism mutation D923N of the Na+, K+-ATPase alpha3 isoform disrupts Na+ interaction at the third Na+ site.

Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific alpha3-isoform of Na(+), K(+)-ATPase. Here, we present the functional consequences of RDP mutation D923N. Relative to the wild type, the mutant exhibits a remarkable approximately 200-fold reduction of Na(+) affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E(1) form with intracellular Na(+). This is the largest effect on Na(+) affinity reported so far for any Na(+), K(+)-ATPase mutant. D923N also affects the interaction with extracellular Na(+) normally driving the E(1)P to E(2)P conformational transition backward. However, no impairment of K(+) binding was observed for D923N, leading to the conclusion that Asp(923) is specifically associated with the third Na(+) site that is selective toward Na(+). The crystal structure of the Na(+), K(+)-ATPase in E(2) form shows that Asp(923) is located in the cytoplasmic half of transmembrane helix M8 inside a putative transport channel, which is lined by residues from the transmembrane helices M5, M7, M8, and M10 and capped by the C terminus, recently found involved in recognition of the third Na(+) ion. Structural modeling of the E(1) form of Na(+), K(+)-ATPase based on the Ca(2+)-ATPase crystal structure is consistent with the hypothesis that Asp(923) contributes to a site binding the third Na(+) ion. These results in conjunction with our previous findings with other RDP mutants suggest that a selective defect in the handling of Na(+) may be a general feature of the RDP disorder.

The C terminus of Na+,K+-ATPase controls Na+ affinity on both sides of the membrane through Arg935.

The Na(+),K(+)-ATPase C terminus has a unique location between transmembrane segments, appearing to participate in a network of interactions. We have examined the functional consequences of amino acid substitutions in this region and deletions of the C terminus of varying lengths. Assays revealing separately the mutational effects on internally and externally facing Na(+) sites, as well as E(1)-E(2) conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr(1017) and Tyr(1018), as well as putative interaction partners, Arg(935) in the loop between transmembrane segments M8 and M9 and Lys(768) in transmembrane segment M5, as crucial to Na(+) activation of phosphorylation of E(1), a partial reaction reflecting Na(+) interaction on the cytoplasmic side of the membrane. Tyr(1017), Tyr(1018), and Arg(935) are furthermore indispensable to Na(+) interaction on the extracellular side of the membrane, as revealed by inability of high Na(+) concentrations to drive the transition from E(1)P to E(2)P backwards toward E(1)P and inhibit Na(+)-ATPase activity in mutants. Lys(768) is not important for Na(+) binding from the external side of the membrane but is involved in stabilization of the E(2) form. These data demonstrate that the C terminus controls Na(+) affinity on both sides of the membrane and suggest that Arg(935) constitutes an important link between the C terminus and the third Na(+) site, involving an arginine-pi stacking interaction between Arg(935) and the C-terminal tyrosines. Lys(768) may interact preferentially with the C terminus in E(1) and E(1)P forms and with the loop between transmembrane segments M6 and M7 in E(2) and E(2)P forms.

Roles of transmembrane segment M1 of Na+,K+-ATPase and Ca2-ATPase, the gatekeeper and the pivot.

In this review we summarize mutagenesis work on the structure-function relationship of transmembrane segment M1 in the Na+,K+-ATPase and the sarco(endo)plasmic reticulum Ca2+-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca2+ interaction/occlusion in Ca2+-ATPase and K+ interaction/occlusion in Na+,K+-ATPase. Leu65 of the Ca2+-ATPase and Leu99 of the Na+,K+-ATPase, located at homologous positions in M1, function as gate-locking residues that restrict the mobility of the side chain of the cation binding/gating residue of transmembrane segment M4, Glu309/Glu329. A pivot formed between a pair of a glycine and a bulky residue in M1 and M3 seems critical to the opening of the extracytoplasmic gate in both the Ca2+-ATPase and the Na+,K+-ATPase.

Importance of Leu99 in transmembrane segment M1 of the Na+, K+ -ATPase in the binding and occlusion of K+.

Twenty-six point mutations were introduced into the N-terminal and middle parts of transmembrane segment M1 of the Na+, K+ -ATPase and its cytosolic extension. None of the alterations to charged and polar residues in the N-terminal part of M1 and its cytosolic extension had any major effect on the cation binding properties, thus rejecting the hypothesis that these residues are involved in cation selectivity. By contrast, specific residues in the middle part of M1, particularly Leu(99), were found critical to K+ interaction of the enzyme. Hence, mutation L99A reduced the affinity for K+ activation of E2P dephosphorylation 17-fold, and L99F reduced the equilibrium level of the K+-occluded intermediate [K2]E2 and increased the rate of K+ deocclusion 39-fold, i.e. more than seen for mutation E329Q of the cation-binding glutamate in M4. L99Q affected K+ interaction in yet another way, the equilibrium level of [K2]E2 being slightly increased despite an increased rate of K+ deocclusion, suggesting that the K+ ions leave and enter the occlusion pocket more frequently than in the wild type. L99Q furthermore affected the ability to discriminate between Na+ and K+ on the extracellular side. Our findings can be explained by a structural model in which Leu(99) and Glu(329) interact and cooperate in K+ binding and gating of the K+ sites. The disturbance of K+ interaction in mutants with alteration to Leu(91), Phe(95), Ser(96), or Leu(98) could be a consequence of the roles of these residues in positioning the M1 helix optimally for the interaction between Leu(99) and Glu(329). Phe(95) may serve to stabilize the pivot for movement of M1 through interaction with Ile(287) in M3.

Mutation of Gly-94 in transmembrane segment M1 of Na+,K+-ATPase interferes with Na+ and K+ binding in E2P conformation.

The importance of Gly-93 and Gly-94 in transmembrane segment M1 of the Na+,K+-ATPase for interaction with Na+ and K+ was demonstrated by functional analysis of mutants Gly-93-Ala and Gly-94-Ala. In the crystal structures of the Ca2+-ATPase, the corresponding residues, Asp-59 and Leu-60, are located exactly where M1 bends. Rapid kinetic measurements of K+-induced dephosphorylation allowed determination of the affinity of the E2P phosphoenzyme intermediate for K+. In Gly-94-Ala, the K+ affinity was reduced 9-fold, i.e., to the same extent as seen for mutation of the cation-binding residue Glu-329. Furthermore, Gly-94-Ala showed strongly reduced sensitivity of the E1P-E2P equilibrium to Na+, with accumulation of E2P even at 600 mM Na+, indicating that interaction of E2P with extracellular Na+ is impaired. On the contrary, in Gly-93-Ala, the affinity for K+ was slightly increased, and the E1P-E2P equilibrium was displaced in favor of E1P. In both mutants, the affinity of the cytoplasmically facing sites of E1 for Na+ was reduced, but this effect was relatively small compared with the effects seen for E2P in Gly-94-Ala. Comparison with Ca2+-ATPase mutagenesis data suggests that the role of M1 in binding of the transported ions is universal among P-type ATPases, despite the low sequence homology in this region. Structural modeling of Na+,K+-ATPase mutant Gly-94-Ala on the basis of the Ca2+-ATPase crystal structures indicates that the alanine side chain comes close to Ile-287 of M3, particularly in E2P, thus resulting in a steric clash that may explain the present observations.

Importance of transmembrane segment M1 of the sarcoplasmic reticulum Ca2+-ATPase in Ca2+ occlusion and phosphoenzyme processing.

The functional consequences of a series of point mutations in transmembrane segment M1 of sarcoplasmic reticulum Ca2+-ATPase were analyzed in steady-state and transient kinetic experiments examining the partial reaction steps involved in Ca2+ interaction and phosphoenzyme turnover. Arginine or leucine substitution of Glu51, Glu55, or Glu58, located in the N-terminal third of M1, did not affect these functions. Arginine or leucine substitution of Asp59, located right at the bend of M1 seen in the crystal structure of the thapsigargin-bound form, caused a 10-fold increase of the rate of Ca2+ dissociation toward the cytoplasmic side. Mutation of Leu60 to alanine or proline and of Val62 to alanine also enhanced Ca2+ dissociation, whereas an 11-fold reduction of the rate of Ca2+ dissociation was observed upon alanine substitution of Leu65, thus providing evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of occluded Ca2+ from its membranous binding sites. Moreover, phosphoenzyme processing was affected by some of the latter mutations, in particular leucine substitution of Asp59, and alanine substitution of Leu65 accelerated the transition to ADP-insensitive phosphoenzyme and blocked its dephosphorylation, thus demonstrating that this part of M1, besides being important in Ca2+ interaction, furthermore, is a critical element in the long range signaling between the transmembrane domain and the cytoplasmic catalytic site.

Mutagenesis of residues involved in control of the Ca2+ entry pathway and conformational changes associated with Ca2+ binding in the SR Ca2+-ATPase.

Rapid kinetic measurements were used to study the rate of Ca(2+) dissociation from the high-affinity Ca(2+) sites of the dephosphoenzyme (i.e., from the E(1)Ca(2) form toward the cytoplasmic side) as well as the rate of Ca(2+) binding with associated conformational changes (E(2) --> E(1)Ca(2) transition) in the wild type and mutants of the sarcoplasmic reticulum Ca(2+)-ATPase expressed in mammalian cells. Cluster mutations as well as single mutations in transmembrane segment M3 resulted in conspicuous effects on the rate of Ca(2+) migration. Furthermore, mutation of Asp(59) in transmembrane segment M1 to arginine exerted a profound effect on Ca(2+) interaction. The data demonstrate an important role for M3 residues in control of the Ca(2+) entry pathway and provide functional evidence in support of a close relationship between this pathway and the water-accessible channel leading between transmembrane segments M1 and M3 in the thapsigargin stabilized E(2) structure. In addition, rapid kinetic measurements demonstrated that the hydrogen bond network involving Asp(813) of loop L6-7 and Lys(758) of M5 is important for the E(2) --> E(1)Ca(2) transition.