The dynamic relationships between the three events that release individual Na + ions from the Na + /K + -ATPase

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1 Received 7 Sep 2011 Accepted 9 Jn 2012 Pulished 14 Fe 2012 DOI: /ncomms1673 The dynmic reltionships etween the three events tht relese individul N + ions from the N + /K + -ATPse Dvid C. Gdsy 1,, Frncisco Beznill 1,, Roert F. Rkowski 1,, Pul De Weer 1, & Miguel Holmgren 1, N + /K + pumps move net chrge through the cell memrne y mediting unequl exchnge of intrcellulr N + nd extrcellulr K +. Most chrge moves during trnsitions tht relese N + to the cell exterior. When pumps re constrined to ind nd relese only N +, memrne voltge-step redistriutes pumps mong conformtions with zero, one, two or three ound N +, therey trnsiently generting current. By pplying rpid voltge steps to squid gint xons, we previously identified three components in such trnsient currents, with distinct relxtion speeds: fst (which nerly prllels the voltge-jump time course), medium speed (τ m = ms) nd slow (τ s = 1 10 ms). Here we show tht these three components re tightly correlted, oth in their mgnitudes nd in the time courses of their chnges. The correltions revel the dynmics of the conformtionl rerrngements tht relese three N + to the exterior (or sequester them into their inding sites) one t time, in n oligtorily sequentil mnner. 1 Mrine Biologicl Lortory, Woods Hole, Msschusetts 02543, USA. Present ddresses: Lortory of Crdic/Memrne Physiology, Rockefeller University, 1230 York Avenue, New York, NY 10065, USA (D.C.G.); Deprtment of Biochemistry nd Moleculr Biology, University of Chicgo, GCIS Building, Room W244, 929 E. 57th Street, Chicgo, Illinois 60637, USA (F.B.); Deprtment of Physiology, University of Pennsylvni, School of Medicine, 3700 Hmilton Wlk, Phildelphi, Pennsylvni 19104, USA (P.D.W.); Moleculr Neurophysiology Section, Porter Neuroscience Reserch Center, Ntionl Institute of Neurologicl Disorders nd Stroke (NINDS), Ntionl Institutes of Helth (NIH), 35 Convent Dr., Building 35 Room 3B-1016, Bethesd, MD 20892, USA (M.H.). Decesed 19 Ferury Correspondence nd requests for mterils should e ddressed to D.C.G. (emil: gdsy@rockefeller.edu) or to M.H. (emil: holmgren@ninds.nih.gov).

2 nture communictions DOI: /ncomms1673 The N + /K + -ATPse ion pump, memer of the P-type fmily (nmed for their phosphorylted intermedites), hrnesses the energy from hydrolysis of one ATP to lterntely export three N + nd import two K + ginst their electrochemicl grdients. By performing this ctive trnsport, the N + /K + pump hs n essentil role in the homeostsis of intrcellulr N + nd K +, crucil to sustining cell excitility, volume nd N + -dependent secondry trnsport. On the sis of iochemicl dt ccumulted during the decde following its discovery 1, the N + /K + -ATPse ws proposed to trnsport N + nd K + ions ccording to model known s the Post-Alers scheme 2,3 (Fig. 1). As ions re trnsported through the N + /K + pump, they ecome temporrily occluded within the protein, inccessile (symolized y prentheses) from either cytoplsmic or extrcellulr side of the memrne, efore eing relesed 4 7. By working in the sence of intrcellulr nd extrcellulr K +, ut presence of intrcellulr ATP, therey effectively restricting N + /K + pumps to the reversile trnsitions ssocited with deocclusion/ occlusion nd extrcellulr relese/inding of N + (conformtions encompssed y the dotted ox in Fig. 1), Nko nd Gdsy 8 were le to detect prestedy-stte electricl signls ccompnying those trnsitions in response to steps of memrne potentil. The signls rise ecuse N + ions trverse frction of the trnsmemrne electric field s they enter or leve their inding sites deep within the pump At given memrne potentil nd externl sodium concentrtion ([N + ] o ), the popultions of pumps with empty inding sites, nd those with ound or occluded N + (tht is, dotted ox conformtions, Fig. 1), rech stedy-stte distriution. A sudden chnge of memrne voltge then shifts the N + -inding equilirium, nd initites redistriution of the pump popultions towrds new stedy-stte rrngement. The consequent chnge in N + -inding-site occupncy cuses N + ions to trvel etween the extrcellulr environment nd the pump interior, through the electric field, so generting current. As the new stedy distriution is pproched, fewer N + ions move, nd the current declines. The electricl signls therefore pper s trnsient currents. By exploiting the ility of xil current delivery in the squid gint xon to very rpidly step memrne voltge, we previously identified three phses of relxtion of trnsient pump currents 11 : fst (comprle to the voltge-jump time course), medium speed (τ m = ms) nd slow (τ s = 1 10 ms). We suggested tht ech phse reflects distinct N + -inding (or relese depending on voltge chnge direction) event with its ssocited conformtionl trnsition (occlusion or deocclusion). Now, mrked technicl dvnces hve llowed us to follow the mounts of chrge moved in ll three phses, simultneously, with unprecedented temporl resolution. We show tht, over rod rnge of [N + ] o nd memrne potentil, regrdless of when the voltge-step ssy is initited, the mount of chrge moved y the fst component is dictted y the sttus of the medium-speed component t tht time, which is itself determined y the extnt sttus of the slow component. Thus, under ll conditions exmined, s N + -ound nd unound pump popultions redistriute, the mounts nd time courses of the three chrge movements re closely correlted. These strict correltions elucidte the dynmic reltionships etween the successive steps tht ind or relese the three N + ions t the N + /K + pump s extrcellulr surfce. Results Pump-medited chrge movement. We otined N + /K + pump current s the component of memrne current inhiited y µm dihydrodigitoxigenin (H 2 DTG), specific nd reversile squid N + /K + pump inhiitor 14 tht, similr to other crdiotonic steroids, immoilizes N + /K + pumps in P-E 2 (N 2 ) stte 15. Figure 2 (middle trce) shows H 2 DTG-sensitive (tht is, pump-medited) current, determined in n xon exposed to 25 mm [N + ] o nd P-E 2.N 3 P-E 2 P-E 2.K 2 (N 3 )E 1 -P E 2 (K 2 ) N 3.E 1 -ATP Extrcellulr E 1.ATP Intrcellulr K 2.E 1.ATP Figure 1 Post-Alers digrm of the N + /K + trnsport cycle. In this simplified scheme, E 1 conformtions represent those in which inding sites my e ccessed from the intrcellulr side. In these sttes, two K + (ornge lls) re relesed, three N + (green lls) re ound nd occluded, nd the protein is phosphorylted. In P-E 2 conformtions, the inding sites ecome ccessile from the externl environment, nd the three N + re relesed, two K + re ound nd occluded, nd the protein utodephosphoryltes. The sttes enclosed y the dotted ox were isolted y removl of K + from ll solutions nd y the presence of 5 mm MgATP (nd of ADP scvengers) 8,14 in the internl solution. 0 [K + ] o, in response to voltge protocol (top trce) tht comprised sequence of seven voltge jumps, from 0 to 120 mv for vrying durtions. Ech step to 120 mv elicited lrge fst component of pump-medited chrge movement (Q f ) of the sme mgnitude, verifying tht ll seven voltge steps egn from the sme initil conditions, tht is, stedy stte t 0 mv. Q f relxtion t 120 mv ws only slightly slower thn the memrne voltge chnge, consistent with Q f reflecting N + ions moving from the ulk solution though prt of the memrne s electric field to their inding sites deep in the protein (Fig. 1; top left). Almost immedite return of the memrne potentil to 0 mv (for exmple, fter the seventh, riefest, step to 120 mv) cused fst pump-medited chrge movement of similr size nd speed, ut opposite sign, consistent with dischrge ck to the ulk solution of ll N + tht hd just entered pump inding sites t 120 mv. But, during the longer voltge steps (for exmple, the first in the sequence), the rpid initil chrge movement t 120 mv ws eventully followed y much slower, nd lower-mplitude, current decy representing the slow chrge component (Q s ) linked to the mjor occlusion trnsition P-E 2.N 3 (N 3 )E 1 -P (Fig. 1). The longest steps to 120 mv llow the distriution of pumps to ttin new stedy stte, poised towrds the occluded conformtion (N 3 )E 1 -P. In those cses, return of the memrne potentil to 0 mv elicited not only rpidly decying, though reltively low mplitude, outwrd Q f, reflecting relese of ound N + from P-E 2.N 3,

3 nture communictions DOI: /ncomms1673 ARTICLE 0 mv 120 mv [N + ] o = 25 mm H 2 DTG-sens 2 µa 10 ms 25 ms Time control c 1 µa 0.5 µa 10 ms 1 ms 0.5 µa s m f Figure 2 Pump-medited trnsient currents t 25 mm [N + ] o. () Voltge protocol, H 2 DTG-sensitive current nd time control. The voltge protocol (upper trce) comprised seven voltge steps from 0 to 120 mv, with durtions of 25, 2, 0.5, 10, 1, 5 nd 0.1 ms. Pump-medited current (middle trce) ws determined y sutrcting memrne current otined in the presence of H 2 DTG from the current record cquired 40 s erlier, efore the inhiitor ws dded. The time control record (lower trce) reflects ny chnges in memrne current during the 40-s period immeditely preceding H 2 DTG ppliction. Arrowheds (lso in,c) indicte zero current. () Pump-medited trnsient current on return to 0 mv t the end of the first (25-ms) voltge step. The red re denotes Q s chrge. (c) Time xis expnsion of. The cyn re represents Q m chrge. These two components were quntified from the time constnts nd mplitudes of extrpolted zero-time intercepts of two-exponentil fit egun 500 µs fter the voltge step ck to 0 mv. Sutrcting this fit from the entire trnsient current yielded Q f chrge (white re). ut lso susequent slowly decying Q s, reflecting deocclusion nd relese through the electric field of N + tht hd ecome occluded in (N 3 )E 1 -P t 120 mv. Becuse, on return to 0 mv, N + ions occluded t 120 mv must wit deocclusion efore they cn e relesed to the ulk solution, the quntity of N + relesed immeditely (Q f ) showed strong inverse correltion with step durtion; the longer the sty t 120 mv, the smller the mplitude of the rpidly decying current on return to 0 mv (Fig. 2). We showed previously tht such pump-medited prestedy-stte currents comprise t lest three components 11, redily distinguished nd seprted when the voltge is stepped ck to 0 mv (Fig. 2,c). After the long sojourn t 120 mv, y fr, the lrgest proportion of the chrge (re underneth the trnsient current trce) ws crried y the slow component (Fig. 2, red re; fitted time constnt t 0 mv, τ s = 8.40 ± 0.01 ms), which ws preceded y t lest two fsterdecying components (Fig. 2c, cyn nd white res; see lso Fig. 3). The slower of these, the medium-speed component (Q m, cyn; fitted time constnt t 0 mv, τ m = 0.55 ± 0.03 ms), ws considerly ( > 10- fold) slower thn the chnge in xon memrne potentil, wheres the decy of the fst component of pump chrge (Q f, white) ws comprle with the speed of the voltge jump (given y the slowest prt of the memrne cpcitnce current decy; τ = 28.0 ± 0.3 µs; Supplementry Fig. S1). Temporl correltions etween the three chrge components. We interpreted 11 the three components of trnsient pump current s (N P-E 2 (N 2 ). 3 )E 1 -P N P-E 2 (N). N P-E 2. N Figure 3 Distinct chnges of the three chrge components nd scheme symolizing inding/relese nd occlusion/deocclusion of three externl N + y the N + /K + pump. () Superposition of the current decys t 0 mv following ech of the seven voltge steps in Figure 2, fter ligning the trces to the initil jump to 120 mv; for clrity, the first 0.1 ms of ll records, nd ll currents < 0, hve een omitted from the figure. With longer stys t 120 mv, Q s (red) incresed monotoniclly, wheres Q m (cyn) nd Q f (white) initilly incresed ut then declined. () Externl N + ions (green) re modelled s intercting with the pump in three distinct, sequentil, inding/relese events ech ssocited with conformtionl trnsition tht rte limits the corresponding chrge component, slow (red), medium speed (cyn) or fst (lck). representing movements of the three N + in nd out of the N + /K + - ATPse (Fig. 3), ech rte-limited y linked occlusion/deocclusion trnsition. Such model predicts tht chnges in the three components on shifting from one stedy-stte distriution to nother must e correlted. To test tht prediction, we hve now trcked the quntity of chrge crried y ech individul component s the durtion of the 120 mv step ws prolonged (Figs 2 nd 3). The progression from the initil stedy stte t 0 mv towrds the finl stedy stte t 120 mv ws followed y ssying, fter vrious times spent t 120 mv (100 µs 25 ms in the experiment of Figs 2 nd 3), the quntity of ech chrge component tht moved ck during reestlishment of the originl stedy stte t 0 mv. The mounts of slow- nd medium-speed chrge, Q s nd Q m, were determined s products of the time constnts nd mplitudes (the time zero intercepts) derived from two-exponentil fits to the current decying t 0 mv, the fits eginning µs fter the step ck to 0 mv. To determine the mount of Q f chrge, ech two-exponentil fit ws sutrcted from its corresponding complete trnsient pump current record, nd the reminder (white res enclosed y current trces in Figs. 2c nd 3) ws numericlly integrted, nd then corrected for ny time-control error; exmples of these smll errors re seen in the time-control trces of Figure 2 nd Supplementry Figure S2. The sizes of the three components of chrge ssyed t 0 mv fter ech visit to 120 mv were ll normlized to the Q s -vlue otined following the longest sty there, nd the results were plotted ginst the time spent t 120 mv (Fig. 4 c). As evident in the current trces of Figure 3 nd in the grph of Figure 4 (which summrize dt from single xon), the

4 nture communictions DOI: /ncomms1673 Q s Norm Q m Norm c Q f Norm [N + ] o = 25 mm Step durtion (ms) Figure 4 Chrge movements mesured in single xon t 0 mv nd 25 mm [N + ] o report on time courses nd sequences of events t 120 mv. () Q s incresed monotoniclly towrds mximum vlue with the durtion of the preceding sty t 120 mv; the mgnitude of Q s fter the 25-ms sty t 120 mv ws used to normlize other chrge components. The solid red line shows n exponentil fit to Q s with τ s = 8.9 ± 0.4 ms. () Q m incresed quickly with fitted τ m = 1.2 ± 0.1 ms (cyn curve) efore decying with time course tht ws well descried y τ s (here fixed t 8.9 ms). (c) Q f developed very rpidly ( < 0.5 ms) nd then decyed (lck curve) with iexponentil time course well descried y time constnts τ m nd τ s (here fixed t 1.2 nd 8.9 ms, respectively). mgnitude of Q s (red) oserved t 0 mv rose mono-exponentilly with time spent t 120 mv, yielding τ s of 8.9 ± 0.4 ms (Fig. 4), chrcteristic of the occlusion trnsition, P-E 2 (N 2 ) N (N 3 ) E 1 -P (Fig. 3, left), t 120 mv fter the third N + hs ound 11. Q m (cyn) from the sme xon exhiited iphsic time course (Figs 3 nd 4), initilly incresing with time spent t 120 mv with τ m = 1.2 ± 0.1 ms, ut then decying long time course tht ws well fit y τ s (8.9 ms). Q f (white re, lck fit curve) from the sme experiment lso displyed iphsic time course (Figs 3 nd 4c), ut with rpid increse followed y iexponentil decy whose time constnts corresponded to τ m (1.2 ms) nd τ s (8.9 ms). Interestingly, the mounts of Q m nd Q f never exceeded smll frction of Q s (t 25 mm [N + ] o, pek Q m nd Q f verged 7 ± 1 nd 14 ± 1% (n = 8), respectively, of the lrgest Q s ), nd were even smller fter ner stedy stte ws reched t 120 mv (stedy Q m mounted to only 4 ± 1%, nd Q f to 3 ± 1% (n = 8) of Q s fter the longest step). Dependence of the three chrge components on extrcellulr N +. Incresing [N + ] o ccelerted the scending phse of ll three components (Fig. 5,), providing support for the interprettion tht ech component reflects distinct N + inding/relese event. As shown for 25 mm [N + ] o ove (Fig. 4), in oth 50 mm (Fig. 5) nd 400 mm [N + ] o (Fig. 5), the time course of the decy of Q m ws well descried y τ s. Similrly, t ech [N + ] o, the decy of Q f ws iexponentil with time constnts corresponding to the respective τ m nd τ s. In ddition, the reltive mplitudes of Q m nd Q f chrge movements on jumping ck to 0 mv fter ttining ner stedy stte t 120 mv, s proportion of their pek mgnitudes fter shorter stys there, were smller t higher [N + ] o (Fig. 5; Supplementry Fig. S2). Discussion Full mechnistic understnding of the series of conformtionl trnsitions tht underlie lternte inding nd trnsport of N + nd K + ions y the N + /K + pump (Fig. 1) requires comintion of structurl informtion t high sptil resolution nd dynmic informtion t high temporl resolution. To otin precise kinetic informtion, the trnsport cycle must e restricted to trctle suset of trnsitions. In this work, we limited pumps to the extrcellulr N + relese/inding trnsitions (dotted ox in Fig. 1) y working in the sence of internl nd externl K +, ut presence of intrcellulr ATP. Previous results estlished tht most of the chrge movement, nd hence voltge dependence, of the trnsport cycle rises within these trnsitions 8,16,17 s N + ions trvel to nd from their inding sites long nrrow ccess chnnels through prt of the electric field 9 11,18. Here we studied the reltionships etween the individul relese/inding trnsitions of extrcellulr N + in ntive N + /K + pumps of internlly dilysed squid gint xons, which llow oth intr- nd extrcellulr environments to e controlled relily, nd memrne voltge to e chnged rpidly. We previously identified three phses of chrge movements during interctions of extrcellulr N + with squid xon N + /K + pumps 11, fst, medium speed nd slow, ech of which we suggested reflected inding or relese of one of the three N +. We found reciprocl reltionship etween the sizes of the medium speed nd slow components tht suggested tht those events occurred in sequence. But our mesurements lcked the precision to discern whether the fst component lso occurred in sequence with the medium-speed component. It remined possile, therefore, tht two N + re relesed/ound sequentilly, ut tht movement of the third N + represents kineticlly distinct, prllel process. By improving our xon superfusion nd dt cquisition systems, we hve now een le to estlish the temporl reltionships nd solute mgnitudes of chnges in ll three components (for exmple, Fig. 3), t the sme time, with previously unttinle precision. We found tht the mount of Q s mesured t 0 mv developed monotoniclly towrds mximum s the preceding step to negtive potentils ws prolonged (Figs 3, 4 nd 5). At ll [N + ] o, the time constnt of tht rise, τ s (verging 7.9 ± 0.3 (n = 7), 5.6 ± 0.2 (n = 7) nd 1.5 ± 0.1 (n = 12) ms, respectively, t 25, 50 nd 400 mm [N + ] o ) mirrored the slow relxtion of the trnsient current (verge τ of 7.9 ± 0.5 (n = 13), 5.5 ± 0.2 (n = 6) nd 1.3 ± 0.1 (n = 10) ms, respectively) t the negtive conditioning potentil ( 120 or 140 mv; 120 mv illustrted here), rther thn the slow current relxtion t 0 mv (verge τ of 9.7 ± 0.3 (n = 14), 9.4 ± 0.3 (n = 7) nd 5.8 ± 0.2 (n = 13) ms, respectively). As the slowly decying current t negtive voltges reports ccumultion of the occluded conformtion, (N 3 )E 1 -P, following inding of the third N + (refs 8 13 nd 16), we cn conclude tht (N 3 )E 1 -P occupncy determines the quntity of Q s oserved t 0 mv, which thus reflects

5 nture communictions DOI: /ncomms1673 ARTICLE [N + ] o = 50 mm 0.15 Q s norm Q m norm Q f norm [N + ] o = 400 mm 0.12 Q s norm Q m norm Q f norm Step durtion (ms) Figure 5 [N + ] o dependence of the time courses nd mplitudes of the three chrge components. Colour nd symol coding, s well s ses for solid line fits, re s in Figure 4. () Dt from single xon t 50 mm [N + ] o : fitted τ s = 6.0 ± 0.2 ms, τ m = 1.5 ± 0.3 ms. () A different xon t 400 mm [N + ] o : fitted τ s = 1.1 ± 0.1 ms, τ m = 0.65 ± 0.10 ms. relese of the first N + from P-E 2 (N 2 ) N, rte-limited y the deocclusion trnsition, (N 3 )E 1 -P P-E 2 (N 2 ) N (Fig. 3, left). In contrst to this monotonic trjectory for Q s, the development of oth Q m nd Q f (ssyed, similr to Q s, t 0 mv) s function of time spent t the negtive conditioning voltge ws iphsic. In oth cses, fter initilly incresing, the chrge eventully diminished with time course tht mtched τ s, indicting tht sequestrtion of pumps in the occluded (N 3 )E 1 -P stte precluded their contriuting to Q m or Q f chrge on return to 0 mv. The events generting Q m nd Q f re therefore ech in series with the occlusion/deocclusion step tht gives rise to Q s. The mono-exponentil relxtion of Q m with time constnt equivlent to τ s rgues tht the Q m -generting trnsition lies, kineticlly, djcent to the (N 3 )E 1 -P P-E 2 (N 2 ) N step. The dditionl fct tht Q f relxed iexponentilly with time constnts tht reflect τ m nd τ s rgues tht the trnsition ssocited with Q f is djcent to tht generting Q m. We cn conclude, then, tht the trnsition tht genertes Q m is sndwiched etween the trnsition ssocited with Q s nd tht ssocited with Q f. Given the three seprle chrge components, the three distinct time constnts, nd the expecttion tht three N + ecome occluded t negtive potentils, the most prsimonious scheme to ccount for these results is tht shown in Figure 3. This model depicts strictly sequentil inding or relese of one N + t time (coloured verticl rrows) through frction of the memrne s electric field, ech N + ion giving rise to trnsient current component with time course governed (coloured horizontl rrows) y the susequent occlusion step in the cse of negtive voltge jumps, or y the preceding deocclusion in the cse of positive voltge jumps. According to this scheme, step from negtive potentil to 0 mv will elicit fst, medium speed nd slow outwrd trnsient chrge components with mgnitudes dictted, respectively, y the occupncies of sttes P-E 2 (N), P-E 2 (N 2 ) nd (N 3 )E 1 -P t the instnt the step is initited. It is the chnges in occupncies of those sttes during the time spent t 120 mv tht re mirrored in the curves in Figures 4 nd 5. The sensitivity to [N + ] o of the time courses of ll three chrge components (Figs 4 nd 5), nd hence of the occupncies of ll three sttes, further supports the existence of three seprte externl N + -inding steps. If ech component indeed reflects motion of single N + ion through prt of the electric field on entering or leving its inding site, the reltively smll 9 pek mgnitudes of Q m nd Q f only 7 14% of the lrgest Q s, over the full mm rnge of [N + ] o exmined cn now e confidently ttriuted to weker electrogenicity of the lst two N + - ion relese steps; this likely reflects the reltively wide extrcellulr ccess pthwy, nd hence wekened electric field, expected t lest for the P-E 2 stte, from which the finl N + ion is relesed, sed on the E 2 BeF 3 C 2 + -ATPse crystl structure 19,20. These findings shed new light on the stepwise structurl chnges tht progressively open the extrcellulr ccess pthwy in the N + /K + pump, model P-type ATPse. Methods Solutions. Gint xons from the squid Loligo pelei were internlly dilysed nd externlly superfused t C with solutions devised to distriute N + /K + pumps mong N + deocclusion/relese trnsitions (Fig. 1, enclosed y dotted ox). The intrcellulr solution contined (in mm): 80 N-HEPES, 57 N-methyld-glucmine-HEPES, 50 glycine, 50 phenylpropyltriethyl-mmonium sulphte (to lock potssium chnnels), 5 dithiothreitol, 2.5 BAPTA, 15 Mg-HEPES, 5 Tris-ATP, 5 phospho(enol) pyruvte tri-n + slt nd 5 phospho-l-rginine mono-n + slt (ph 7.6, djusted with HEPES). The composition of the extrcellulr solution ws (in mm): 400 N-sulphmte, 75 C 2 + -sulphmte, 1 3,4-diminopyridine, tetrodotoxin, 5 Tris-HEPES nd 0.05 EDTA (ph 7.7, djusted with N-methyl-dglucmine or sulphmic cid). N-sulphmte ws replced y tetrmethylmmonium-sulphmte in solutions contining lower externl N + concentrtions. Osmollity of ll solutions ws ~930 mosmol kg 1. In principle, these experimentl solutions could support uncoupled N + efflux 21, non-cnonicl mode of N + /K + pump opertion tht occurs severl orders of mgnitude more slowly thn the slowest N + occlusion/deocclusion step, nd so could not ffect our mesurements. Electrophysiology. Voltge steps were generted nd currents recorded using 14-it high-speed AD/DA conversion ord (A4D1, Innovtive Integrtion) with softwre developed in-house. Currents were filtered t khz with n 8-pole Bessel filter (Frequency Devices 90IP), then smpled t 1 MHz. N + /K + pump current ws resolved s current sensitive to H 2 DTG, specific nd reversile inhiitor of the squid pump 14. Pump currents were nlysed with in-house softwre nd OriginL v8. To study the temporl correltions etween the individul components of pump-medited current, the experimentl system must remin nerly perfectly stle. Becuse pump currents re otined y sutrcting memrne current trces cquired efore nd fter H 2 DTG ddition, ny ltertion of other electricl properties of the system during tht time intervl would preclude meningful interprettion of the sutrcted current. For exmple, chnges in series resistnce, presumly due to chnges in the Frnkenhäuser-Hodgkin spce surrounding the xon, re known to occur over time 22. So we redesigned our superfusion system to speed the ction of H 2 DTG (τ now ~7 s; Supplementry Fig. S3) nd time-control errors re now diminished, s evident from the time control-trces similr to those in Figure 2 nd Supplementry Figure S2. An dditionl technicl improvement is more powerful cquisition system tht llows n entire experiment to e

6 nture communictions DOI: /ncomms1673 cptured in single record while mintining the high temporl resolution of 1 MHz smpling. References 1. Skou, J. C. The influence of some ctions on n denosine triphosphtse from peripherl nerves. Biochim. Biophys. Act. 23, (1957). 2. Alers, R. W. Biochemicl spects of ctive trnsport. Annu. Rev. Biochem. 36, (1967). 3. Post, R. L., Kume, T., Toin, T., Orcutt, B. & Sen, A. K. Flexiility of n ctive center in sodium-plus-potssium denosine triphosphtse. J. Gen. Physiol. 54, (1969). 4. Glynn, I. M., Hr, Y. & Richrds, D. E. The occlusion of sodium ions within the mmmlin sodium-potssium pump: its role in sodium trnsport. J. Physiol. 351, (1984). 5. Beugé, L. A. & Glynn, I. M. Occlusion of K ions in the unphosphorylted sodium pump. Nture 280, (1979). 6. Glynn, I. M. & Krlish, S. J. Occluded ctions in ctive trnsport. Annu. Rev. Biochem. 59, (1990). 7. Post, R. L., Hegyvry, C. & Kume, S. Activtion y denosine triphosphte in the phosphoryltion kinetics of sodium nd potssium ion trnsport denosine triphosphtse. J. Biol. Chem. 247, (1972). 8. Nko, M. & Gdsy, D. C. Voltge dependence of N trnsloction y the N/K pump. Nture 323, (1986). 9. Hilgemnn, D. W. Chnnel-like function of the N,K pump proed t microsecond resolution in gint memrne ptches. Science 263, (1994). 10. Holmgren, M. & Rkowski, R. F. Chrge trnsloction y the N + /K + pump under N + /N + exchnge conditions: intrcellulr N + dependence. Biophys. J. 90, (2006). 11. Holmgren, M. et l. Three distinct nd sequentil steps in the relese of sodium ions y the N + /K + -ATPse. Nture 403, (2000). 12. Rkowski, R. F. Chrge movement y the N/K pump in Xenopus oocytes. J. Gen. Physiol. 101, (1993). 13. Peluffo, R. D. Effect of ADP on N + -N + exchnge rection kinetics of N, K-ATPse. Biophys. J. 87, (2004). 14. Rkowski, R. F., Gdsy, D. C. & De Weer, P. Stoichiometry nd voltge dependence of the sodium pump in voltge-clmped, internlly dilyzed squid gint xon. J. Gen. Physiol. 93, (1989). 15. Stürmer, W. & Apell, H. J. Fluorescence study on crdic glycoside inding to the N,K-pump. Ouin inding is ssocited with movement of electricl chrge. FEBS Lett. 300, 1 4 (1992). 16. Heyse, S., Wuddel, I., Apell, H. J. & Stürmer, W. Prtil rections of the N, K-ATPse: determintion of rte constnts. J. Gen. Physiol. 104, (1994). 17. Stürmer, W., Bühler, R., Apell, H. J. & Läuger, P. Chrge trnsloction y the N,K-pump: II. Ion inding nd relese t the extrcellulr fce. J. Memr. Biol. 121, (1991). 18. Gdsy, D. C., Rkowski, R. F. & De Weer, P. Extrcellulr ccess to the N, K pump: pthwy similr to ion chnnel. Science 260, (1993). 19. Olesen, C. et l. The structurl sis of clcium trnsport y the clcium pump. Nture 450, (2007). 20. Toyoshim, C., Norimtsu, Y., Iwsw, S., Tsud, T. & Ogw, H. How processing of sprtylphosphte is coupled to lumenl gting of the ion pthwy in the clcium pump. Proc. Ntl Acd. Sci. USA 104, (2007). 21. Glynn, I. M. in The Enzymes of Biologicl Memrnes Vol. 3 (ed. Mrtonosi, A.N.) (Plenum, 1985). 22. Stimers, J. R., Beznill, F. & Tylor, R. E. Sodium chnnel gting currents. Origin of the rising phse. J. Gen. Physiol. 89, (1987). Acknowledgements This reserch ws directly supported y the Intrmurl Reserch Progrm of the Ntionl Institutes of Helth (NIH), NINDS, grnts NIH HL36783 to D.C.G., nd NIH U54GM nd R01GM to F.B. We thnk the Section on Instrumenttion of the Ntionl Institute of Mentl Helth for custom hrdwre. Author contriutions D.C.G., F.B., R.F.R., P.D.W. nd M.H. designed nd performed experiments. D.C.G., F.B., P.D.W. nd M.H. nlysed the dt. D.C.G. nd M.H. wrote the mnuscript. All uthors discussed the results during the execution of the project. Additionl informtion Supplementry Informtion ccompnies this pper t nturecommunictions Competing finncil interests: The uthors declre no competing finncil interests. Reprints nd permission informtion is ville online t reprintsndpermissions/ How to cite this rticle: Gdsy, D.C. et l. The dynmic reltionships etween the three events tht relese individul N + ions from the N + /K + -ATPse. Nt. Commun. 3:669 doi: /ncomms1673 (2012). License: This work is licensed under Cretive Commons Attriution-NonCommercil- NoDerivtive Works 3.0 Unported License. To view copy of this license, visit cretivecommons.org/licenses/y-nc-nd/3.0/