New Insights into the Role of the N Terminus in Conformational Transitions of the Na,K-ATPase

The deletion of 32 residues from the N terminus of the α1 catalytic subunit of the rat Na,K-ATPase (mutant α1M32) shifts the E1/E2 conformational equilibrium toward E1, and the combination of this deletion with mutation E233K in the M2-M3 loop acts synergistically to shift the conformation further toward E1 (Boxenbaum, N., Daly, S. E., Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998)J. Biol. Chem. 273, 23086–23092). To delimit the region of the cytoplasmic N terminus involved in these interactions, the consequences of a series of N-terminal deletions of α1 beyond Δ32 were evaluated. Criteria to assess shifts in conformational equilibrium were based on effects of perturbation of the entire catalytic cycle ((i) sensitivity to vanadate inhibition, (ii) K+ sensitivity of Na-ATPase measured at micromolar ATP, (iii) changes in K′ATP, and (iv) catalytic turnover), as well as estimates of the rates of the conformational transitions of phospho- and dephosphoenzyme (E1P → E2P and E2(K+) → E1 + K+). The results show that, compared with α1M32, the deletion of up to 40 residues (α1M40) further shifts the poise toward E1. Remarkably, further deletions (mutants α1M46, α1M49, and α1M56) reverse the effect, such that these mutants increasingly resemble the wild type α1. These results suggest novel intramolecular interactions involving domains within the N terminus that impact the manner in which the N terminus/M2-M3 loop regulatory domain interacts with the M4-M5 catalytic loop to effect E1 ↔ E2 transitions. The deletion of 32 residues from the N terminus of the α1 catalytic subunit of the rat Na,K-ATPase (mutant α1M32) shifts the E1/E2 conformational equilibrium toward E1, and the combination of this deletion with mutation E233K in the M2-M3 loop acts synergistically to shift the conformation further toward E1 (Boxenbaum, N., Daly, S. E., Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998)J. Biol. Chem. 273, 23086–23092). To delimit the region of the cytoplasmic N terminus involved in these interactions, the consequences of a series of N-terminal deletions of α1 beyond Δ32 were evaluated. Criteria to assess shifts in conformational equilibrium were based on effects of perturbation of the entire catalytic cycle ((i) sensitivity to vanadate inhibition, (ii) K+ sensitivity of Na-ATPase measured at micromolar ATP, (iii) changes in K′ATP, and (iv) catalytic turnover), as well as estimates of the rates of the conformational transitions of phospho- and dephosphoenzyme (E1P → E2P and E2(K+) → E1 + K+). The results show that, compared with α1M32, the deletion of up to 40 residues (α1M40) further shifts the poise toward E1. Remarkably, further deletions (mutants α1M46, α1M49, and α1M56) reverse the effect, such that these mutants increasingly resemble the wild type α1. These results suggest novel intramolecular interactions involving domains within the N terminus that impact the manner in which the N terminus/M2-M3 loop regulatory domain interacts with the M4-M5 catalytic loop to effect E1 ↔ E2 transitions. The Na,K-ATPase or sodium pump is a ubiquitous integral membrane protein that catalyzes the glycoside sensitive, ATP-coupled exchange of three intracellular Na+ for two extracellular K+ ions across the plasma membrane of all animal cells. The sodium pump is essential to the maintenance of the electrochemical gradients of Na+ and K+ across the cell membrane, providing the driving force for the transport of nutrients into the cell and maintaining the cellular resting membrane potential. It comprises two essential subunits: a large catalytic α subunit (∼100 kDa), containing the ligand binding and phosphorylation sites and a smaller, highly glycosylated β subunit (∼35–55 kDa), which acts as a chaperone for α (for review see Refs. 1Sweadner K.J. Biochim. Biophys. Acta. 1989; 988: 185-220Crossref PubMed Scopus (868) Google Scholar and 2Lingrel J.B. Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar). A third subunit, γ (∼7 kDa), was found in the kidney where it functions as a regulator of the pump (see Refs. 3Mercer R.W. Biemesderfer D. Bliss Jr., D.P. Collins J.H. Forbush B.3 rd. J. Cell Biol. 1993; 121: 579-586Crossref PubMed Scopus (184) Google Scholar and 4Therien A.G. Blostein R. Am. J. Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar). The sodium pump is a member of a family of transporters known as P-type ATPases that are directly phosphorylated and dephosphorylated on a conserved aspartate residue within their cytoplasmic domain during the course of the reaction cycle. Both the phosphorylated and dephosphorylated forms of the enzyme can exist in at least two states that undergo conformational transitions (E1P → E2P and E2 → E1) that are coupled to the ion-translocating steps. Definitive evidence for distinct E1 and E2 conformational states and a role of the N terminus in effecting E1 ↔ E2transitions was first obtained in 1975 by Jorgensen (5Jorgensen P.L. Biochim. Biophys. Acta. 1975; 401: 399-415Crossref PubMed Scopus (242) Google Scholar, 6Jorgensen P.L. Biochim. Biophys. Acta. 1977; 466: 97-108Crossref PubMed Scopus (92) Google Scholar) using tryptic cleavage of the renal enzyme in the presence of different ligands. Later confirmatory evidence was obtained using N-terminal deletion mutants expressed in cultured cells (7Daly S.E. Lane L.K. Blostein R. J. Biol. Chem. 1994; 269: 23944-23948Abstract Full Text PDF PubMed Google Scholar). This study showed that whereas deleting up to and including the lysine-rich cluster is without effect, removal of 32 residues (mutant α1M32) alters the enzyme kinetics by shifting the E1/E2conformational equilibrium toward E1 forms. Interestingly, a similar shift toward E1 is caused by Glu233→ Lys substitution in the first (M2-M3) cytoplasmic loop. Furthermore, the combined removal of the N-terminal 32 residues and replacement of Glu233 → Lys (mutant α1M32E233K) results in a remarkably synergistic shift in poise toward E1state(s) as evidenced in analyses of several characteristic properties including, for examples, extraordinary insensitivity to vanadate, high affinity for ATP (5 μm), and slow (≤500 min−1) catalytic turnover of α1M32E233K. These findings were interpreted to indicate that interactions between these two cytoplasmic regions and the M4-M5 catalytic loop are critical for conformational coupling (8Boxenbaum N. Daly S.E. Javaid Z.Z. Lane L.K. Blostein R. J. Biol. Chem. 1998; 273: 23086-23092Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The present study was carried out to define more precisely the role of the N terminus in conformational transitions. Accordingly, we investigated the consequences of further deletions of the N terminus of the Na,K-ATPase beyond the first 32 residues to near the beginning of the first transmembrane segment, using several criteria to assess shifts in the steady-state E1 ↔ E2 poise. The results of this analysis reveal a progressively increased shift in the E1/E2 poise toward E1, followed by reversal toward the wild type α1 E1/E2 distribution. Combined with secondary structure predictions of the N terminus, this behavior identifies a self-regulatory domain within the N terminus of the Na,K-ATPase that modulates conformational transitions via novel intramolecular interactions. Predictions of the secondary structure of the N-terminal sequence were evaluated using the following algorithms: PREDATOR (9Frishman D. Argos P. Prot. Engin. 1996; 9: 133-142Crossref PubMed Scopus (357) Google Scholar), Sspro (10Baldi P. Brunak S. Frasconi P. Soda G. Pollastri G. 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Putative helical regions were then ascribed to those sequences for which the above algorithms concur on helicity. The desired mutations were introduced into the 5′SacI230-SalI875restriction fragment cassette of the rat α1 cDNA as described previously (7Daly S.E. Lane L.K. Blostein R. J. Biol. Chem. 1994; 269: 23944-23948Abstract Full Text PDF PubMed Google Scholar). The mutant cassettes were then ligated into the rat α1 cDNA in the place of the wild typeSacI-SalI cassette. The full-length mutant cDNAs were released form the shuttle vector by digestion withHindIII, ligated into the expression plasmid pCDNA3.1 (Invitrogen), and orientation of the cDNA was determined by restriction analysis. HeLa cells were transfected with the pCDNA-α1 mutant constructs using either the calcium phosphate method (18Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: PubMed Scopus Google Scholar) or the (Invitrogen), and cells the rat α1 and mutants were as described previously J.B. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, L.K. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). HeLa cells the mutant α1 were in and as described previously (7Daly S.E. Lane L.K. Blostein R. J. Biol. Chem. 1994; 269: 23944-23948Abstract Full Text PDF PubMed Google Scholar). The above was with see J.B. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), which was several were from the mutant cells as described J.B. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, L.K. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). Protein was determined with a Methods Enzymol. PubMed Scopus Google Scholar). Na,K-ATPase was measured as the of from as previously described R. Methods Enzymol. PubMed Scopus Google Scholar). and the were for at with all The reaction was by the of for Na,K-ATPase were and was to in and of Na,K-ATPase were carried out using ATP to to ATP and sensitivity of of the cultured cells (see Refs. L.K. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Daly S.E. J.B. Blostein R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, and S.E. Lane L.K. Blostein R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Na-ATPase was measured at ATP as described previously (7Daly S.E. Lane L.K. Blostein R. J. Biol. Chem. 1994; 269: 23944-23948Abstract Full Text PDF PubMed Google Scholar), with of and to and was determined with 40 of vanadate were to the and with the to the Na,K-ATPase obtained at vanadate and expressed as of that obtained in the of vanadate, were by the to a using a analysis of a as described A. D. Am. J. Physiol. 1978; PubMed Google Scholar). were determined as described S.E. Lane L.K. Blostein R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). were with and for at to HeLa The was then with either for at or with for (see was then carried out for at in a of in and were determined by with and and the enzyme in the of The of → E1 + was measured by the of E1 from E2(K+) as described S.E. Lane L.K. Blostein R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), with the described by and Blostein A.G. Blostein R. Am. J. Physiol. 1999; PubMed Google Scholar). of in the presence of high Forbush B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), the of → E2P was determined by the of of following of the of → of to of E2P Forbush B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, B. Biochemistry. PubMed Scopus Google Scholar). Accordingly, the enzyme was first phosphorylated in containing to and with for at to was then by with a containing of ATP, and which the to were for of for of up to were obtained by the to for least two different membrane obtained from at least two different were The are of at least three is the of To more precisely the and the role of the cytoplasmic N terminus of the α1 catalytic subunit of the Na,K-ATPase on the steady-state E2 conformational we first the of the secondary structure of this N-terminal This region is a highly structure with a high of helicity. A of the results of several analyses (see the presence of three domains residues and To or these wild type HeLa cells were transfected with N-terminal deletion mutants of the α1 subunit of the rat Na,K-ATPase to either or of the first and and and or of the of the mutant constructs enzyme of HeLa cell in criteria were to shifts in the E1/E2 that the of the catalytic cycle and that to the E1 ↔ E2 and ↔ E2P transitions. The sensitivity of Na,K-ATPase to by vanadate, (ii) of Na-ATPase to to the E2(K+) → → E1 + (iii) catalytic and (iv) to affinity ATP binding to The of K+ → → E2 + (see (ii) and of → E2P as in with the wild type α1 the poise in E1 ↔ E2 of the deletion mutant is toward E1 forms of the enzyme (7Daly S.E. Lane L.K. Blostein R. J. Biol. Chem. 1994; 269: 23944-23948Abstract Full Text PDF PubMed Google Scholar, S.E. Lane L.K. Blostein R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). To into the effects of further deletions on conformational we investigated the effect of vanadate on Na,K-ATPase is a of phosphate that to P-type ATPases in the Jr., L. J. Biol. Chem. 1978; Full Text PDF PubMed Google Scholar). sensitivity of enzyme to by vanadate is a of the of enzyme in the E2 by the in and in and are to vanadate is that the E1/E2 equilibrium of these progressively shifts toward E1 as up to 40 residues are from the N this shift is to a more wild sensitivity by deleting residues (mutants α1M46, α1M49, and of behavior of Na,K-ATPase mutants during steady-state from the in of K+ on with as of without from in turnover was determined as the of Na,K-ATPase measured at and ATP to measured at in the presence of and (see are the of the of in are of the of in different from different from different from different from different from different from different from different from different from different from different from different from from the in with as of without from in turnover was determined as the of Na,K-ATPase measured at and ATP to measured at in the presence of and (see are the of the of in are of the of in different from in a micromolar ATP to the high affinity phosphorylation the of Na-ATPase to K+ is a to in the K+ of the reaction cycle → → E1 + which these (7Daly S.E. Lane L.K. Blostein R. J. Biol. Chem. 1994; 269: 23944-23948Abstract Full Text PDF PubMed Google Scholar, C. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). as previously and in K+ the Na-ATPase of α1 that of The deletion of 40 results in up to Interestingly, further α1M46, α1M49, and K+ These in suggest a shift in the E1/E2conformational equilibrium toward E1 removal of up to 40 residues from the N terminus, which is by deleting The catalytic turnover of the Na,K-ATPase is as the to the measured at in the presence of ATP, and to the enzyme in the (see M. Biophys. PubMed Scopus Google Scholar). the catalytic turnover of α1 and the mutant previously in with a shift in the E1 ↔ E2 poise toward E1. a that of α1. deletion of residues the catalytic turnover to near that of α1. in K+ sensitivity of Na-ATPase at ATP suggest a in the of a in the K+ of the reaction which can by a (see in Biochim. Biophys. Acta. 1994; PubMed Scopus Google Scholar). ATP first with affinity by to the K+ followed by → → + K+). the the slow of K+ from E2(K+) via E2(K+) → → E1 + K+ is followed by high affinity ATP binding to E1. Accordingly, a mutation a in K+ of Na-ATPase at micromolar ATP a in a in of the that and for affinity ATP binding compared with α1 (see in and similar to that of the wild type α1 with the reversal of the K+ effect on Na-ATPase at It was that the of is that that of α1 this enzyme is by K+ at This characteristic of is further in the series of we compared the slow of K+ from E2(K+) via the E2(K+) → → E1 + K+ for of the in S.E. Lane L.K. Blostein R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A.G. Blostein R. Am. J. Physiol. 1999; PubMed Google Scholar) we the slow of from E2(K+) is at following of E2(K+) in Na+ containing at that the phosphorylation of the of is present to this is a of E1 from in the changes in E2(K+) from the in well to The for and in that of α1. are similar to previously for α1 and α1M32, S.E. Lane L.K. Blostein R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This that for and the E2(K+) ↔ ↔ E1 + is to the the for α1M46, α1M49, and are similar or those for and that deletion of residues the above to a more wild E1/E2 distribution. It that whereas the K+ sensitivity of Na-ATPase at ATP turnover via of K+ with the of A as the course of of E1 from E2(K+) via B. It is known changes in of the two are K+ of the Na-ATPase is for compared with their similar rates of E2(K+) → K+ of and are similar to that of their rates of E2(K+) → E1 are that of α1. these and the that this reaction was at the of followed by rates of E2(K+) → E1 with is of conformational rates of Na,K-ATPase are of the of in from were from the different from different from different from different from different from different from are of the of in from were from the different from in a To the of the conformational of the to the E1 ↔ E2 shifts we the → E2P at as previously described Forbush B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, B. Biochemistry. PubMed Scopus Google Scholar). Accordingly, the enzyme is first phosphorylated by at high to is then by a in to with the of and the of of E2P is that of the of E2P from the course of the → in and in compared with the wild type α1 the of this is for and α1M46, α1M49, and similar or rates to the wild type these reaction analyses of the mutants and wild type enzyme the behavior in of the catalytic cycle in The sequences of the N and are the least conserved domains the P-type ATPases found in and the and animal These regions that pump as well as binding for regulatory that pump by these sequences Biochim. Biophys. Acta. 1998; PubMed Scopus Google Scholar). the plasma membrane which domains in N terminus in or in terminus in J.F. B. R. J.F. H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, S. P. PubMed Scopus Google Scholar, A. J. 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Javaid Z.Z. Lane L.K. Blostein R. J. Biol. Chem. 1998; 273: 23086-23092Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, S.E. Lane L.K. Blostein R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). we show that or (α1M40) of results in a shift toward E1 forms of the show that and or of well as the shift to a more wild E1 ↔ The changes the behavior of is and α1M49, a all three mutants resemble wild type α1 with to the vanadate (ii) sensitivity to K+ at ATP, (iii) of E1 from and (iv) of → These indicate or of structure that with beyond the first residues a of the N terminus with which effects of the conformational such as increased of to the binding into cytoplasmic that with the N terminus were first obtained by Jorgensen and Collins P.L. Collins J.H. Biochim. Biophys. Acta. PubMed Scopus Google Scholar) between the N terminus and cytoplasmic a study (8Boxenbaum N. Daly S.E. Javaid Z.Z. Lane L.K. Blostein R. J. Biol. 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A is the N terminus, which is by domain A. the N terminus is a from the transmembrane the first helical and that which a different sequence from that of the The present study a role of the N terminus of the The in a region of the N terminus that acts as domain the E1/E2 conformational transitions. the of the wild type enzyme helical region can with either interactions in the E2 the M2-M3 loop and the catalytic loop to can with a domain of the M2-M3 loop in E1 to it from the catalytic loop as in the E1 is or or see interacts with the M2-M3 a on the the and the E1 The is a shift in the conformational equilibrium in of E1. the and or of (see the on the M2-M3 loop it to with the large catalytic M4-M5 the wild type α1 It is that the and of the catalytic subunit of the rat Na,K-ATPase similar helical in their N Furthermore, of the first of and (mutants and S. Daly, and R. Blostein, shift the conformational equilibrium for enzyme toward E1 that the N terminus of the Na,K-ATPase domain present in all three we domain within the N terminus of the sodium which modulates conformational novel intramolecular interactions within the cytoplasmic domains of the are to further the residues in the N terminus that are involved in these interactions. Z. Javaid for of the and constructs and for and critical of the The of is

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