Fluorometric Measurements of Intermolecular Distances between the α- and β-Subunits of the Na+/K+-ATPase

The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the α-subunit, which mediates ATP hydrolysis and ion translocation, as well as the β-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the α- and β-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the α- and β-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the α-subunit and the β-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the β-subunit in relation to the α-subunit and suggest that the α- and β-subunits move toward each other during the E2 to E1 conformational transition. The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the α-subunit, which mediates ATP hydrolysis and ion translocation, as well as the β-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the α- and β-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the α- and β-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the α-subunit and the β-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the β-subunit in relation to the α-subunit and suggest that the α- and β-subunits move toward each other during the E2 to E1 conformational transition. The Na+/K+-ATPase is a ubiquitous protein complex that is required for maintaining the concentration gradients of Na+ and K+ across the plasma membrane (1Kaplan J.H. Annu. Rev. Biochem. 2002; 71: 511-535Crossref PubMed Scopus (875) Google Scholar, 2Jørgensen P.L. Hakansson K.O. Karlish S.J. Annu. Rev. Physiol. 2003; 65: 817-849Crossref PubMed Scopus (433) Google Scholar). This complex is comprised of two mandatory subunits. The α-subunit is the major catalytic subunit, and the β-subunit is required for proper trafficking of the complex to the plasma membrane (3Jørgensen P.L. Pedersen P.A. Biochim. Biophys. Acta. 2001; 1505: 57-74Crossref PubMed Scopus (109) Google Scholar, 4Geering K. Theulaz I. Verrey F. Hauptle M.T. Rossier B.C. Amer. J. Physiol. 1989; 257: C851-C858Crossref PubMed Google Scholar, 5Jassier F. Jaunin P. Geering K. Rossier B.C. Horisberger J.D. J. Gen. Physiol. 1994; 103: 605-623Crossref PubMed Scopus (130) Google Scholar). A third subunit, γ, is also present, at least in certain tissues, which, although not required for function, has been identified as a regulatory protein (6Therien A.G. Pu H.X. Karlish S.J. Blostein R. J. Bioenerg. Biomembr. 2001; 33: 407-414Crossref PubMed Scopus (49) Google Scholar). The Na+/K+-ATPase is one member of more than 200 P-type ATPases, so named because the protein is transiently phosphorylated at a highly conserved aspartate residue upon ATP hydrolysis (7Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (746) Google Scholar). A series of crystal structures showing different conformational states of a highly homologous P-type ATPase, the sarcoplasmic reticulum Ca2+-ATPase (SERCA), 4The abbreviations used are: SERCA, sarcoplasmic reticulum Ca2+-ATPase; FRET, fluorescence resonance energy transfer; TMRM, tetramethylrhodamine-6-maleimide; FM, fluorescein-5-maleimide; MOPS, 4-morpholinepropanesulfonic acid; D, donor; A, acceptor.4The abbreviations used are: SERCA, sarcoplasmic reticulum Ca2+-ATPase; FRET, fluorescence resonance energy transfer; TMRM, tetramethylrhodamine-6-maleimide; FM, fluorescein-5-maleimide; MOPS, 4-morpholinepropanesulfonic acid; D, donor; A, acceptor. has provided insight into the structure of the Na+/K+-ATPase as well as the conformational flexibility of the pump during ion translocation (8Toyoshima C. Nakasaki M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1605) Google Scholar, 9Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (805) Google Scholar, 10Toyoshima C. Mizutani T. Nature. 2004; 430: 529-535Crossref PubMed Scopus (380) Google Scholar, 11Toyoshima C. Nomura H. Tsuda T. Nature. 2004; 432: 361-368Crossref PubMed Scopus (380) Google Scholar, 12Sorensen T.L.M. Moller J.V. Nissen P. Science. 2004; 304: 1672-1675Crossref PubMed Scopus (371) Google Scholar). It has been demonstrated through these structures that the SERCA pump undergoes significant conformational changes during ion transport. However, as the SERCA pump is comprised of only the α-subunit, no information is available about the involvement of the β-subunit in the conformational flexibility of the Na+/K+-ATPase. Direct structural information of the β-subunit has been limited to electron microscopy studies (13Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google Scholar, 14Rice W.J. Young H.S. Martin D.W. Sachs J.R. Stokes D.L. Biophys. J. 2001; 80: 2187-2197Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The results from these experiments have proposed a putative location for the β-subunit in relation to the α-subunit. However, this model is inconsistent with some biochemical experiments, and alternative structural models have been proposed (2Jørgensen P.L. Hakansson K.O. Karlish S.J. Annu. Rev. Physiol. 2003; 65: 817-849Crossref PubMed Scopus (433) Google Scholar, 15Hasler U. Crambert G. Horisberger J.D. Geering K. J. Biol. Chem. 2001; 276: 16356-16364Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Furthermore, as the electron microscopy study yields a static representation of the ion pump, it is difficult to elucidate the relative movements of the α- and β-subunits during ion translocation. Voltage clamp fluorometry has been recognized as a powerful technique to monitor the conformational dynamics of ion channels (16Mannuzu L.M. Moronne M.M. Isacoff E.Y. Science. 1996; 271: 213-216Crossref PubMed Scopus (397) Google Scholar, 17Cha A. Bezanilla F. Neuron. 1997; 19: 1127-1140Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 18Cha A. Bezanilla F. J. Gen. Physiol. 1998; 112: 391-408Crossref PubMed Scopus (120) Google Scholar). The method has also been extended to determine distance constraints and the relative movement between protein subunits that accompany gating of the Shaker potassium channel and the human glutamate transporter using fluorescence resonance energy transfer (FRET) (19Koch H.P. Larsson H.P. J. Neurosci. 2005; 25: 1730-1736Crossref PubMed Scopus (100) Google Scholar, 20Glauner K.S. Mannuzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (246) Google Scholar, 21Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar). These studies have demonstrated that small scale movements between subunits accompany channel gating. Our laboratory has previously utilized voltage clamp fluorometry to demonstrate that residues on the α- and β-subunits of the Na+/K+-ATPase can be site-specifically with the tetramethylrhodamine-6-maleimide changes that by changes in membrane ion can be to the two main conformational E1 and E2 of the Na+/K+-ATPase J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar). To examine the between the α- and β-subunits of the we have used voltage clamp fluorometry to determine distance constraints between the α- and β-subunits We have also the relative movement between the α- and β-subunits to the of the plasma membrane as the ion pump shuttles between the two conformational states as described by the Albers-Post E1 and E2 Annu. Rev. Biochem. PubMed Scopus Google Scholar, C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The results from this study have identified the orientation of the β-subunit in relation to the α-subunit and the relative movement of the two subunits during ion transport. and of Na+/K+-ATPase with no residues and J.H. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google and and PubMed Scopus Google for the of the Na+/K+-ATPase as well as the were into as described J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar). were by from Xenopus laevis and were with of α-subunit and of β-subunit which using the were in and MOPS, at for and were for in and and in and to the Na+ concentration J. Gen. Physiol. PubMed Scopus Google Scholar). fluorescence labeling by in fluorescein-5-maleimide for on in the by in labeling experiments, the were in the labeling experiments, the were with and this fluorophore and that each fluorophore with at the it is that ion pump with two residues be at the following and the donor experiments, the be the as the be through the of the However, be a small of ion that with two donor This a and the distance be for under conditions were performed in Na+ and with under were performed in Na+ pump with of Voltage on the of a fluorescence with a were using a voltage clamp by a constant to from a The following were and and and and were from with a to the were by a clamp and were and with and and distance the Na+/K+-ATPase with two residues on the α- and the were with as described previously in this of the with pump and the of donor following The results of were and the were to a some were by a the time constant to the used and the time constant only a small of the K.S. Mannuzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (246) Google Scholar). The of the is However, it be to a on the plasma membrane of the To determine the of energy transfer the following and the time of donor in the presence and the absence of T. Scopus Google Scholar). to distance using the is the distance between the donor and acceptor and is the distance to for a T. Scopus Google Scholar). is by the is the of the donor and acceptor is the donor in absence of the is the of and is the orientation for a G. and Scholar). The of the donor and acceptor on the oocytes, which a and through the of the following is the and is the with to the at each R. J. Biophys. J. Full Text PDF PubMed Scopus Google Scholar, J. of Google Scholar). The two were with to the and To the in the distance to the following were and and is the of FM, is the of TMRM, and is the of each fluorophore and A. Bezanilla F. J. Gen. Physiol. 1998; 112: 391-408Crossref PubMed Scopus (120) Google Scholar, Science. PubMed Scopus Google Scholar). and has that following on the M5-M6 loop of the α-subunit and on the of the β-subunit, the ion pump is and that the can be to the of the Na+/K+-ATPase J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). and of the Na+/K+-ATPase that a on the M3-M4 loop or on the α- and the were for demonstrated by measuring pump upon the of K+ in voltage clamp experiments in the presence of to the Na+/K+-ATPase of the were functional and to the which the results as the also be upon the of be by because of the for the Na+/K+-ATPase used in these studies J.D. J. Physiol. 2002; Scopus Google Scholar). However, the be to the Na+/K+-ATPase PubMed Scopus Google Scholar). labeling of residues of the Na+/K+-ATPase has previously provided insight into the conformational dynamics of the ion pump during the the fluorescence can be to the conformational of the ion pump and the of fluorescence can be with ion across the plasma membrane J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar). Our is to investigate the relative conformational dynamics between the α- and β-subunits of the ion pump by labeling subunits with and changes in Our to determine significant between two or two β-subunits of the ion pump in the Xenopus plasma membrane. The results from this two it insight into the of the Na+/K+-ATPase in the plasma and it determine the of distance constraints between the α- and β-subunits be as a between two or two on the loop between and on the on the loop between and on the and to the on the were for It has previously been that the conformational of the ion pump can be following labeling with at each of these during Na+ or J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar, G. T. Biophys. J. 2003; Scholar). These were with the donor fluorophore and the acceptor fluorophore transfer by measuring the in of the acceptor fluorophore following of the donor fluorophore under These conditions of the two main conformational states of the ion pump by the membrane of the M. Nature. PubMed Scopus Google Scholar). these experiments, we not the conformational of the the in fluorescence is the donor fluorophore is to the acceptor experiments have that following labeling of and changes in fluorescence were during in membrane two residues with and and the a in be during voltage These results from the were with the of the acceptor fluorophore the and were with donor and acceptor The with a of than demonstrated of FRET, the of fluorescence for these to or than of the fluorescence the on the α- and the with a of than fluorescence changes that were than and is only a of energy transfer between or in the The for the is most to the of ion on the which a small of ion to be the in the plasma membrane. the a significant energy transfer and the results from this not the presence of a or structure of the Na+/K+-ATPase in the plasma membrane of the Xenopus it is to between the α- and β-subunits. in and can to in the of for a residues that significant can the of energy transfer between the donor and acceptor and the distance which from irreversible fluorophore J. of Google Scholar). the relative of and using under conditions each of the residues were highly the of the of for the the for the to be the that of the residues a to be A. Bezanilla F. J. Gen. Physiol. 1998; 112: 391-408Crossref PubMed Scopus (120) Google Scholar, H.P. Larsson H.P. J. Neurosci. 2005; 25: 1730-1736Crossref PubMed Scopus (100) Google Scholar, 20Glauner K.S. Mannuzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (246) Google Scholar). for the to be K.S. Mannuzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (246) Google Scholar). Our has demonstrated that residues with fluorescence changes as a of it has the fluorescence changes the of a of the the fluorophore or the of a of the fluorophore J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar, G. T. Biophys. J. 2003; Scholar). To examine the relative orientation of the used to in the E1 and E2 The of and with significant in at of residues with or in the E1 or E2 the in the of fluorescence following is not the of a in The in fluorescence is the of a in of the determine distance constraints between the α- and ion that two one on the α- and the were with the donor and acceptor or To determine between two the time of donor following labeling with the donor fluorophore in the presence or absence of the acceptor The of the donor fluorophore on the donor a the not to acceptor has a and on the at a (16Mannuzu L.M. Moronne M.M. Isacoff E.Y. Science. 1996; 271: 213-216Crossref PubMed Scopus (397) Google Scholar). The of the donor fluorophore is in the presence of acceptor fluorophore to energy transfer, which the on a to more and which yields a Furthermore, that demonstrate a of the donor fluorophore in the presence of the acceptor fluorophore with a of residues that K.S. Mannuzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (246) Google Scholar, 21Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, J. of Google Scholar). The between the of the irreversible of the donor fluorophore in the presence and absence of the acceptor fluorophore can be used to the distance between the two using the T. Scopus Google Scholar). The of of the donor in the presence or absence of acceptor at a membrane to a constant distance between subunits for and only with a of than were used for and to a were through the by the of donor significant in the time of donor for or that were with and the acceptor fluorophore to the time of the donor in the presence or absence of the acceptor fluorophore can be used to the distance constraints between the α- and β-subunits. the two of residues in this the were to be for and for A and of the α- and during the of distance we were in the relative movement of the α- and β-subunits during the E2 to E1 by Na+ and K+ concentration However, to the on the donor only structural changes be using this alternative that with a fluorophore have been identified because the fluorescence changes during the E1 to E2 and the fluorescence is by of J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar). it has been that the fluorescence changes on the β-subunit to the conformational changes of the although it has not been to determine the relative movement of subunits. However, in experiments, the of demonstrate no fluorescence changes during the E1 to E2 of the ion pump, and these have not been J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar). The absence of fluorescence for these has two the residue is not to the and be or the residue is with a fluorophore and is no during the E1 to E2 transition. to residues that were with a fluorophore were to the E1 to E2 and to changes in following fluorophore labeling of two of these To residues that to the E1 to E2 on were one residue is to a fluorophore fluorescence changes during that the E2 to E1 and it is the residue a fluorophore no fluorescence changes during To investigate between these the were with donor and acceptor these experiments, we not measuring the relative movement of the two one is used as a to transfer energy to the have a fluorophore one be to a fluorescence that is the of the of at one residue It is which of the two residues has a donor or acceptor fluorophore as each the on the other only one of the two residues is no in fluorescence be this the of and were has that the and functional and not demonstrate a in fluorescence during the E1 to E2 following labeling with J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar, G. T. Biophys. J. 2003; Scholar). laboratory has that and demonstrate fluorescence changes during the E1 to E2 J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar, G. T. Biophys. J. 2003; Scholar). of these were with the donor and the acceptor and a significant in acceptor following donor upon the of and the in fluorescence upon the of that the fluorescence is to the Na+/K+-ATPase it is that and to fluorophore labeling that the at these residues to the E1 to E2 transition. the relative movement of the α- and β-subunits be using these residues the fluorescence is to the E1 to E2 transition. the between the of previously by irreversible donor to this method be highly to changes in between the two labeling with the donor and the acceptor for the in acceptor following that the two residues toward each a in acceptor that the two residues from each in that the relative distance of the two residues is and were functional and following the of a small in acceptor and This is to the Na+/K+-ATPase as the is following the of these that is a conformational between the α- and β-subunits during the E2 to E1 transition. these experiments is that the in fluorescence is the of a small of between two as to between two subunits of one ion However, it has been that the of between two ion is and the in fluorescence be the of the two subunits toward each other during the E2 to E1 transition. is that the in is to a in the orientation of the donor or acceptor fluorophore between the E1 and E2 and not to a in the distance between the two However, as is no significant in for or at of the residues in Na+ or K+ the in is most to a in distance between the α- and β-subunits. The results from this study information on the relative orientation and movement of the α- and β-subunits of the Na+/K+-ATPase during ion transport. through the electron microscopy studies of (13Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google and with the SERCA pump, it proposed that the β-subunit the it has been that this model not in the A study of the β-subunit proposed that at least two of the β-subunit the of the U. Crambert G. Horisberger J.D. Geering K. J. Biol. Chem. 2001; 276: 16356-16364Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). it has been that the β-subunit and the can be R. Karlish J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the model proposed by Maunsbach and (13Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google only one of the β-subunit to the α-subunit and a distance between the of the α-subunit and the β-subunit, alternative structure proposed the β-subunit is to and to (2Jørgensen P.L. Hakansson K.O. Karlish S.J. Annu. Rev. Physiol. 2003; 65: 817-849Crossref PubMed Scopus (433) Google Scholar, 15Hasler U. Crambert G. Horisberger J.D. Geering K. J. Biol. Chem. 2001; 276: 16356-16364Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, R. Karlish J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). this the between the β-subunit and M3-M4 and M5-M6 and in the structure proposed by (13Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google the and the from with the structure by (13Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google the β-subunit is to the It is to that of the distance have been in a model by that the residues in the to the membrane. the M3-M4 and the M5-M6 only residues and the residue from the β-subunit is proposed to be to the plasma membrane to it that the residues on the in relation to the plasma membrane. residues at different from the membrane the the and the residues in model with a this is to be the in for the β-subunit to be to the M3-M4 and M5-M6 be which, as not during these experiments is that each which the at each at However, it is that one fluorophore at a than the the acceptor fluorophore at a than the donor fluorophore at one or the of ion that with two acceptor the of ion that with one donor and one acceptor or with two donor the ion not donor be no significant in the the donor fluorophore at a the relative of with and a be This into a distance than the this is the the model not be the model (2Jørgensen P.L. Hakansson K.O. Karlish S.J. Annu. Rev. Physiol. 2003; 65: 817-849Crossref PubMed Scopus (433) Google Scholar). a of the electron microscopy it has been proposed that the ion pump a complex as were in the electron microscopy studies between these experiments, it that the β-subunit of two were in However, experiments not this as no significant is between β-subunits and A between two also in the electron microscopy study (13Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google Scholar). this the which is to the is to the of a ion However, the results from experiments not to or this to the static of the electron microscopy studies can be used to insight into the relative movement of the α- and β-subunits during ion translocation. following of the ion pump, it that the of the β-subunit undergoes a conformational during the E2 to E1 J.H. PubMed Scopus Google Scholar). However, the relative movement of the α- and β-subunit during ion translocation residues to the membrane of the fluorescence is to the conformational dynamics of the one can study the relative movement of residues on the transporter following labeling with donor and acceptor It that a small in between the α- and β-subunits is during the E2 to E1 transition. This small significant in that the α- and β-subunits toward each other during the E2 to E1 transition. Furthermore, the results from the experiments that the in fluorescence during voltage or is not by changes in orientation of the fluorophore J.H. T. U. A. 2003; PubMed Scopus Google Scholar, T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar, G. T. Biophys. J. 2003; Scholar). We have previously that the on the β-subunit results in in for Na+ T. J. Gen. Physiol. 2005; PubMed Scopus Google Scholar). is conserved in to or residues on the human and we proposed that between for the of ion The results from the study demonstrate that is a between the α- and β-subunits. it be that the relative movement of the α- and β-subunits be by or residues on the β-subunit. This be the by which different have different on ion from of distance we have been to differentiate between two of the β-subunit in relation to the α-subunit and have that is a structural between the of the β-subunit and the M3-M4 and the M5-M6 of the α-subunit during ion translocation. This study a by which small distance changes of membrane can be on by We F. for and G. for experiments as well as and for