Functional Role of Oxygen-Containing Residues in the Fifth Transmembrane Segment of the Na,K-ATPase α Subunit

doi:10.1006/abbi.1999.1124

Archives of Biochemistry and Biophysics

Volume 364, Issue 2, 15 April 1999, Pages 254-263

https://doi.org/10.1006/abbi.1999.1124 Get rights and content

Abstract

The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembrane segment of the Na, K-ATPase α subunit were studied using site-directed mutagenesis, expression, and kinetics analysis. Nonconservative replacements Thr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover. Replacements at these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-ATPase activity (in the absence of K⁺). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na⁺and K⁺affinities (K_1/2for Na,K-ATPase activation). Thus, the high Na-ATPase activity does not appear related to cation-binding alterations. It is probably associated with conformational alterations which lead to an acceleration of enzyme dephosphorylation by Na⁺acting at the extracellular space (Argüelloet al. J. Biol. Chem.271, 24610–24616, 1996). Nonconservative substitutions at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease of enzyme turnover. Enzyme–Na⁺interaction was greatly changed in these enzymes, while their activation by K⁺did not appear affected. Although the Na⁺K_1/2for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala,n_Hill= 0.75; Tyr771Ser,n_Hill= 0.92; Control,n_Hill= 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na⁺dependence (n_Hill= 1.91). All Tyr771-substituted enzymes showed low steady-state levels of phosphoenzyme during Na-activated phosphorylation by ATP. Phosphorylation levels were not increased by oligomycin, although the drug bound and inactivated Tyr771-substituted enzymes. No E1 ↔ E2 equilibrium alterations were detected using inhibition by vanadate as a probe. The data suggest that Tyr771 might play a central role in Na⁺binding and occlusion without participating in K⁺–enzyme interactions.

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Neurological disease mutations of α3 Na+,K+-ATPase: Structural and functional perspectives and rescue of compromised function
2016, Biochimica et Biophysica Acta - Bioenergetics
Na⁺,K⁺-ATPase creates transmembrane ion gradients crucial to the function of the central nervous system. The α-subunit of Na⁺,K⁺-ATPase exists as four isoforms (α1–α4). Several neurological phenotypes derive from α3 mutations. The effects of some of these mutations on Na⁺,K⁺-ATPase function have been studied in vitro. Here we discuss the α3 disease mutations as well as information derived from studies of corresponding mutations of α1 in the light of the high-resolution crystal structures of the Na⁺,K⁺-ATPase. A high proportion of the α3 disease mutations occur in the transmembrane sector and nearby regions essential to Na⁺ and K⁺ binding. In several cases the compromised function can be traced to disturbance of the Na⁺ specific binding site III. Recently, a secondary mutation was found to rescue the defective Na⁺ binding caused by a disease mutation. A perspective is that it may be possible to develop an efficient pharmaceutical mimicking the rescuing effect.
The rapid-onset dystonia parkinsonism mutation D923N of the Na +,K+-ATPase α3 isoform disrupts Na+ interaction at the third Na+ site
2010, Journal of Biological Chemistry
Citation Excerpt :
In support of our hypothesis, mutation of Thr771, Ser772, and Tyr768 has led to quite significant (10- to 20-fold) reductions of the apparent Na+ affinity (38, 41, 47, 48). Importantly the effect was selective for Na+ in mutants T771A (38, 41, 48), Y768L (47), and Y768F (49), consistent with an involvement in the binding of the third Na+ ion. For mutant S772A, both Na+ and K+ affinities have been reported significantly reduced relative to wild type (48, 50, 51).
Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific α3-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 ∼200-fold reduction of Na⁺ affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E₁ 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 E₁P to E₂P conformational transition backward. However, no impairment of K⁺ binding was observed for D923N, leading to the conclusion that Asp⁹²³ is specifically associated with the third Na⁺ site that is selective toward Na⁺. The crystal structure of the Na⁺,K⁺-ATPase in E₂ form shows that Asp⁹²³ 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 E₁ form of Na⁺,K⁺-ATPase based on the Ca²⁺-ATPase crystal structure is consistent with the hypothesis that Asp⁹²³ 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.
Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na+ transport, and Gln-923 (M8) is essential for Na,K-ATPase activity
2005, Journal of Biological Chemistry
Citation Excerpt :
The data shown in Table I (compare both a and b with c and d of T774A) are consistent with the report that T774A reduced the apparent affinity for Na+, as estimated by Na+-dependent phosphorylation without affecting the affinity of Tl+ (K+) binding in a yeast expression system (37). The hydroxyl group of Tyr-771 has also been reported to be important for binding Na+ without participating in K+-enzyme interaction (42). The direct coordination of Gln-923 to the first Na+ and the first K+ without coordination from Thr-774 to any Na+ and K+ has been proposed based on homology modeling (46).
The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K_0.5 values for Na⁺, as estimated by the Na⁺-dependent phosphoenzyme formation (K_0.5^Na,EP) or Na,K-ATPase activity (K_0.5^Na,ATPase), were increased by around 2∼8-fold in the case of T774A, V920E, and E954A. The apparent K_0.5 values for K⁺, as estimated by the Na,K-ATPase (K_0.5^K,ATPase) or p-nitrophenylphosphatase activity (K_0.5^K,pNPPase), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K_0.5^K,ATPase. The apparent K_0.5 values for ATP (K_0.5^EP), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6∼2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K_0.5^ATPase) and ATP-induced inhibition (K_i0.5^pNPPase) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na⁺ and/or K⁺ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na⁺ and also in the high and low affinity ATP effects rather than the transport of K⁺.
Identification of the transmembrane metal binding site in Cu +-transporting P<inf>IB</inf>-type ATPases
2004, Journal of Biological Chemistry
Citation Excerpt :
In the case of CopA, Asn675, Val718, and Asn721 substitutions lead to alterations compatible with the mutant enzymes being “locked” in an E1 conformation and thus largely inactive and undergoing little or none “backdoor” phosphorylation. Significant functional alterations have also been shown by previous work characterizing mutations of conserved polar amino acids located in the equivalent region of the Na,K-ATPase (58), Ca-ATPase (59, 60), and H,K-ATPase (61). Depending on the model protein and the introduced mutation, lack of activity, high activity in the absence of contra-ion (Na-ATPase), enzyme uncoupling, and reduced phosphoenzyme levels have been reported.
P_IB-type ATPases have an essential role maintaining copper homeostasis. Metal transport by these membrane proteins requires the presence of a transmembrane metal occlusion/binding site. Previous studies showed that Cys residues in the H6 transmembrane segment are required for metal transport. In this study, the participation in metal binding of conserved residues located in transmembrane segments H7 and H8 was tested using CopA, a model Cu⁺-ATPase from Archaeoglobus fulgidus. Four invariant amino acids in the central portion of H7 (Tyr⁶⁸² and Asn⁶⁸³) and H8 (Met⁷¹¹ and Ser⁷¹⁵) were identified as required for Cu⁺ binding. Replacement of these residues abolished enzyme activity. These proteins did not undergo Cu⁺-dependent phosphorylation by ATP but were phosphorylated by P_i in the absence of Cu⁺. Moreover, the presence of Cu⁺ could not prevent the enzyme phosphorylation by P_i. Other conserved residues in the H7-H8 region were not required for metal binding. Mutation of two invariant Pro residues had little effect on enzyme function. Replacement of residues located close to the cytoplasmic end of H7-H8 led to inactive enzymes. However, these were able to interact with Cu⁺ and undergo phosphorylation. This suggests that the integrity of this region is necessary for conformational transitions but not for ligand binding. These data support the presence of a unique transmembrane Cu⁺ binding/translocation site constituted by Tyr-Asn in H7, Met and Ser in H8, and two Cys in H6 of Cu⁺-ATPases. The likely Cu⁺ coordination during transport appears distinct from that observed in Cu⁺ chaperone proteins or catalytic/redox metal binding sites.
Electrophysiological Analysis of the Mutated Na,K-ATPase Cation Binding Pocket
2003, Journal of Biological Chemistry
Citation Excerpt :
The stationary current of N776Q (at -20 mV) was comparable with that of the wild-type enzyme. This is in contrast to the enzyme activity found by others, which was 7% (49) and 22% (53) of that of the wild-type activity. This discrepancy is probably due to differences in determining the enzyme activity and/or the expression system.
Na,K-ATPase mediates net electrogenic transport by extruding three Na⁺ ions and importing two K⁺ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp⁷⁷⁶ (Gln, Asp, Ala), Glu⁷⁷⁹ (Asp, Gln, Ala), Asp⁸⁰⁴ (Glu, Asn, Ala), and Asp⁸⁰⁸ (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K⁺-stimulated stationary currents and pre-steady-state currents under electrogenic Na⁺/Na⁺ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na⁺/K⁺ pump currents, the other constructs could be classified according to the [K⁺] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K⁺. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na⁺/Na⁺ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn⁷⁷⁶ and Asp⁸⁰⁴, respectively, decreases the extracellular Na⁺ affinity.
Site-directed mutagenesis of cation coordinating residues in the gastric H, K-ATPase
2001, Archives of Biochemistry and Biophysics
Site-mutations were introduced into putative cation binding site 1 of the H,K-ATPase at glu-797, thr-825, and glu-938. The side chain oxygen of each was not essential but the mutations produced different activation and inhibition kinetics. Site mutations thr-825 (ala, leu) and glu-938 (ala, gln) modestly decreased the apparent affinity to K⁺, while glu-797 (gln) was equivalent to wild type. As expected of competitive inhibition, mutations of thr-825 and glu-938 that decreased the apparent affinity for K⁺ also increased the apparent affinity for SCH28080. This is consistent with the participation of thr-825 and glu-938 in a cation binding domain. The sidechain geometry, but not the sidechain charge of glu-797, is essential to ATPase function as the site mutant glu-797 (gly) inactivated the H,K-ATPase, while glu-797 (gln) was active but the apparent affinity to SCH 28080 was decreased by fourfold. Lys-793, a unique residue of the H,K-ATPase, was essential for ATPase function. Since this residue is adjacent to site 1, the result suggests that charge pairing between lys-793 and residues at or near this site may be essential to ATPase function.

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^☆: This work was supported by National Institute of Health Grant HL28573 (J.B.L.).

^☆☆: De Pont, J. J.

²: J.M.A. is a recipient of a Research Development Award for Minority Faculty, HL 03373, from the National Institute of Health.

³: To whom correspondence should be addressed. Fax: (508) 831-5933. E-mail:[email protected].

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Cited by (17)

Neurological disease mutations of α3 Na<sup>+</sup>,K<sup>+</sup>-ATPase: Structural and functional perspectives and rescue of compromised function

The rapid-onset dystonia parkinsonism mutation D923N of the Na <sup>+</sup>,K<sup>+</sup>-ATPase α3 isoform disrupts Na<sup>+</sup> interaction at the third Na<sup>+</sup> site

Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na<sup>+</sup> transport, and Gln-923 (M8) is essential for Na,K-ATPase activity

Identification of the transmembrane metal binding site in Cu <sup>+</sup>-transporting P<inf>IB</inf>-type ATPases

Electrophysiological Analysis of the Mutated Na,K-ATPase Cation Binding Pocket

Site-directed mutagenesis of cation coordinating residues in the gastric H, K-ATPase

Regular ArticleFunctional Role of Oxygen-Containing Residues in the Fifth Transmembrane Segment of the Na,K-ATPase α Subunit☆,☆☆

Abstract

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Regular Article
Functional Role of Oxygen-Containing Residues in the Fifth Transmembrane Segment of the Na,K-ATPase α Subunit☆,☆☆