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1 912 iophysial Journal Volume 66 Marh Pre-Steady-State Transient Currents Mediated by the Na/K Pump in Internally Perfused Xenopus Ooytes M. Holmgren and R. F. Rakowski Department of Physiology and iophysis, University of Health Sienes, The Chiago Medial Shool, North Chiago, Illinois 664 US STRCT Pre-steady-state transient urrents have been investigated in the vegetal pole of Xenopus ooytes using the open-ooyte vaseline-gap tehnique of Taglialatela, Toro, and Stefani (iophysial Joumal. 61:78-82, 1992). Voltage pulses 4 ms in duration were made from a holding potential of -4 mv to ommand potentials over the range -16 to + 6 mv in inrements of 2 mv. Current reords (averaged 2X; sampled every 2 ps) in the presene of dihydroouabain (DHO) or absene of extemal Na+ (Nao) were subtrated from urrent reords obtained under Na/Na exhange onditions, i.e internally perfused with 5 mm Na+, 5 mm TP, and 5 mm DP (K+-free) and externally superfused with 1 mm Na+,K+-free solution. Transient urrents were dependent on intraellular Na+ and nuleotides, and diminished by ativation of forward pumping; they were also redued by 1 pg ml-1 of oligomyin applied to the external solution. These properties of the pre-steady state urrents are onsistent with the Na/K pump operating in its eletroneutral Na/Na exhange mode. The voltage dependene of the DHOand Nao-sensitive transient urrents was analyzed using a pseudo two-state model in whih only the rate oeffiient for Naobinding/reolusion is voltage-dependent (Rakowski, R.F J. Gen. Physiol. 11: ). The apparent valene of the harge moved during the on (zq-,,) and off (Zqoff) of the pulse were.96 ±.5 and.95 ±.5 for Na-sensitive, and 1.1 ±.7 and.85 ±.6 for DHO-sensitive transient urrents, respetively. The total amount of harge moved (Qtot) and the mid-point voltage of the harge distribution (Vq) were 23 ± 15 pc and ± 5.1 mv, and 268 ± 34 pc and mv for Na- and DHO-sensitive transient urrents, respetively. The apparent valene (zk) and the voltage at whih the forward and bakward rates are equal (Vk) obtained from the relaxation rates were.8 ±.5 and ± 1. mv, and and ± 9. mv for the Na- and DHO-sensitive pre-steady state urrents, respetively. The values of the parameters were not statistially signifiantly different between the Nao- and DHO-sensitive transient urrents. Exluding the first 6 ps after the onset of a voltage step whih was not temporally resolved, transient urrents showed no indiation of a rising phase. These results support the idea that harge transloation ours within an extemal aess hannel at a rate that is governed by a voltage-dependent binding/reolusion proess and a voltage-independent deolusion/unbinding proess. INTRODUCTION Kineti information about the steps in the Na/K pump yle that transloate harge an be obtained from urrent relaxation experiments. The pump, whih is initially in a steady state, is perturbed by a sudden hange of an external parameter suh as substrate onentration or voltage. fter the perturbation the oupany of intermediate states of the enzyme will approah a new steady-state distribution. The presteady-state urrent assoiated with the relaxation gives information on the harge transloating steps of the pump yle (ULuger and pell, 1988; Lauger, 1991). Nakao and Gadsby (1986) reorded strophanthidinsensitive transient urrents in ventriular myoytes in response to imposed voltage jumps. These transient urrents were reorded with the whole-ell path-lamp tehnique using wide-tipped pipettes and K+-free pipette (internal) and external solutions, whih should limit the Na/K pump to operate in its Na/Na exhange mode. Depolarizing voltage jumps from a holding potential of -4 mv produed strophanthidin-sensitive transient outward urrents, and re- Reeivedforpubliation 3 September 1993 and in finalform 21 Deember ddress reprint requests to Dr. R. F. Rakowski at the Department of Physiology and iophysis, University of Health Sienesf/he Chiago Medial Shool, 3333 Green ay Rd., North Chiago, IL 664. Tel.: ; Fax: X 1994 by the iophysial Soiety /94/3/912/11 $2. polarization eliited inwardly direted transient urrents that deayed monoexponentially. The amount of harge transloated (Q), measured as the integral of the transient urrent, showed a sigmoid voltage dependene that ould be desribed by a oltzmann equation with a steepness fator appropriate for a single harge moved through the entire membrane field. The relaxation rate (k, the inverse of the exponential time onstant) approahed a minimum at positive potentials, but inreased steeply when the potential was made more negative. Reently, Rakowski (1993) has studied the voltage dependene of the pre-steady-state harge movement from the endogenous Na/K pump in Xenopus ooytes using a twomiroeletrode voltage lamp tehnique. Dihydroouabain (DHO)- and Nao-sensitive transient urrents measured in K+-free external solutions had the harateristis of membrane harge movement (Chandler et al., 1976): voltagedependent relaxation rate, saturating sigmoid voltage dependene of the quantity of harge moved, and equality of the harge moved at the on and off of the voltage step. The voltage dependene of harge movement by the Na/K pump in Xenopus ooytes was found to be similar to that observed in myoytes by Nakao and Gadsby (1986). We have studied pre-steady-state harge transloation by the Na/K pump working under Na/Na exhange onditions inxenopus ooytes using the open-ooyte vaseline-gap tehnique (Taglialatela et al., 1992). With this tehnique we have been able to explore transient urrents 6 p,s after the onset

2 Holmgren and Rakowski Na/K Pump Transient Currents 913 of the voltage step. Moreover, the tehnique has allowed us to ontrol the intraellular medium and, therefore, study the intraellular substrate dependene of the harge transloation. In K+-free solutions, and in the presene of 5 mm DP, 5 mm TP, 5 mm Na+ in the intraellular solution and 1 mm Na+ in the extraellular solution, a Nao- or DHOsensitive omponent of the transient urrent ould be deteted. These urrents delined toward zero in the steadystate, and were dependent on the presene of intraellular Na+ and nuleotides. The transient urrent was diminished by ativation of forward Na/K pumping or addition of 1,ug ml-' oligomyin. These harateristis strongly suggest that the pre-steady-state urrents are assoiated with the Na/K pump operating in its Na/Na exhange mode. We have found that there is no appreiable differene between the DHO- and Na.-sensitive transient urrents. two-state model in whih only the bakward rate oeffiient is voltagedependent fit the experimental data reasonably well. Two findings support the two-state model: (i) transient urrents resolved after 6,us showed no indiation of a rising phase, and (ii) the apparent valene of the transloated harge alulated either from the steady-state harge distribution or from the urrent relaxation rates were in agreement and approximately equal to 1. However, there is a disrepany between the mid-point voltage Vq measured from the steadystate harge versus voltage relationship and Vk measured from the voltage dependene of the harge relaxation rate that is not predited by a simple two-state model. The values of Vk are muh more negative (--13 mv) than the orresponding values of Vq (--6 mv). The two-state model is, therefore, oversimplified. preliminary report of these findings has been published (Holmgren and Rakowski, 1993b). MTERILS ND METHODS Preparation of ooytes Ooyte-positive, adult female frian lawed toads (Xenopus laevis) were obtained from Xenopus I (nn rbor, MI) and were maintained on a high protein diet in fresh water tanks. The animals were anesthetized by immersion in ie for about 3 min until unresponsive to tatile stimulation. Surgery was performed as desribed by Smith et al. (1991). small inision is made in the posterolateral side of the ventral part of the animal, and the required amount of ovarian tissue is removed. fter suturing, the animals are transferred to a 1-m deep, old water ontainer, whih is allowed to warm up at room temperature for at least 1 h before returning the animal to its tank. nimals that have undergone surgery are kept in a separate tank. When neessary, animals were killed by deapitation after being anesthetized. Ooytes were isolated by treatment of ovarian tissue for h with ollagenase (type I; Sigma, St. Louis, MO, 2 mg ml-') in a modified Ringer solution (Rakowski et al., 1991). Ooytes were inubated at 17 C in either normal arth or K+-free arth solutions (see below) both with 5,ug ml-' of neomyin added. lthough the results did not differ depending on the inubation solution, we have found that the ooytes are in better ondition and an be used for a longer period after surgery when they are inubated in normal arth's solution. Ooytes were usually used from 1-4 days after the ollagenase treatment. Solutions for inubation of ooytes The normal arth's solution had the following omposition (in mm): 88 NaCl, 1 KC1, 2.4 NaHCO3,.82 MgSO4,.33 Ca(NO3)2,.41 CaCl2, and 5 2-amino-2-hydroxymethylpropane-1,3-diol (TRIS)/4-(2-hydroxymethyl)-1-piperazineethanesulfoni aid (HEPES) (ph 7.1). When needed, KCI was omitted in order to obtain K+-free arth solution. Modified Ringer solution ontained the following salts (in mm): 87.5 NaCl, 2.5 KCl, 1. MgCl2, 5. TRIS/HEPES (ph 7.6). Experimental solutions loona OK external solution had the following omposition (in mm): 1 sodium sulfamate, 2 tetraethylammonium sulfamate (TE), 1 MgSO4, 1 TRIS/HEPES (ph 7.6). ONa OK external solution was obtained by equimolar substitution of tetramethylammonium sulfamate for sodium sulfamate. The omposition of the internal solution was varied to find optimal onditions. In most of the experiments, the internal solution ontained (in mm) 5 sodium sulfamate, 2 TE sulfamate, 1 MgSO4, 5 MgTP, 5 Tris-DP, 5 1,2-bis(2-aminophenoxy)ethane-NNN,N',N'-tetraaeti aid, 3N-methyl- D-gluamine (NMG) sulfamate, 1 TRIS/HEPES (ph 7.3). Na+-free internal solution was prepared by equimolar substitution of NMG sulfamate for sodium sulfamate. Experiments were performed in K+- and Cl--free external and internal solutions to minimize non-pump-mediated urrents. bsene of external K+ also prevents the normal forward mode of operation of the Na/K pump. TE was present to redue K-hannel-mediated resting and time-dependent urrents. TP and DP were present to promote eletroneutral Na/Na exhange (DeWeer, 197; Glynn and Hoffman, 1971; Cavieres and Glynn, 1979). oth the internal and external solutions were nominally Ca2l-free to prevent outward Na/Ca exhange urrent and the ativation of Cal'-dependent anioni urrent. Finally, the solutions were Cl--free to redue hloride urrent (Miledi and Parker, 1984). Oligomyin was dissolved in dimethyl sulfoxide (DMSO) at a onentration of 2 mg ml-' and was prepared immediately before eah experiment in whih it was used. ll reagents were purhased from Sigma. The most reent experiments were performed in the presene of 5 mm a(no3)2, 2 mm Ni(NO,)2, 1,uM Gd(NO3)3 in the external solutions. MgSO4 was substituted by magnesium sulfamate. a2' and Ni2+ were used to blok K+ urrent and the Na/Ca exhanger (Rakowski, 1993). Gd3+ was used to blok strethativated ation hannels (Yang and Sahs, 1989). Eletrophysiologial measurements n open-ooyte, guarded-seal tehnique (Taglialatela et al., 1992) was used to investigate pre-steady-state harge movement by the endogenous Na/K pump in the vegetal pole ofxenopus ooytes. The external ompartment is ontinuously superfused by gravity at a rate of -2 ml min-' and aspiration by a vauum pump. Internal perfusion was performed with a annula onneted via polyethylene tubing (.58 mm I.D.) to a syringe pump (SPlOOi; World Preision Instruments, In., Sarasota, FL) that injets intraellular solution at a rate of 3-5,ul h-'. In some experiments, replaement of intraellular solution was required. In order to minimize the dead spae in the perfusion system, a 33G needle -15-mm long was used in the final setion of tubing after a T-juntion. t a rate of 5,lI h-1, the new intraellular solution is expeted to arrive at the ell -5 min after the solution up to the T-juntion is hanged. n additional syringe pump (Sage Instruments; model 355) was used to rapidly lear the solution up to the T-juntion and drive the syringe ontaining the new intraellular solution. The eletroni system was designed by Taglialatela et al. (1992) and purhased from DGN Corporation (Minneapolis, MN; Model C-1 High Performane Ooyte Clamp). The holding potential was usually -4 mv. Voltage pulses 4 ms in duration were made from the holding potential to ommand potentials over the range -16 to + 6 mv in inrements of 2 mv. Pulses were applied every 5 ms. Current reords shown below usually were the result of averaging 2 repetitions of the pulse protool. Data were olleted using a ommerially available analog to digital onverter system (it-1 DM interfae, 1 KHz; xon Instruments, In., Foster City, C) and software (PCLMP version 5.5; xon Instrument, In.) running on an IM ompatible omputer system (Dell Computer Corp., ustin, TX). The analog signal was filtered at 5 KHz before being digitized, and was sampled every 2 ps. n improvement in signal-to-noise ratio ould have been ahieved by using a faster sampling rate or by using a 1-KHz filter ut-off for the sampling rate employed.

3 914 iophysial Journal Transient urrents were alulated by subtration of the urrent reords obtained after halting Na/Na exhange as desribed below from those reords aquired prior to stopping the exhange. Na/Na exhange was stopped by three different means: 1) diret addition of 5,l of 2 mm DHO to the extraellular ompartment (the DHO onentration attained was -2 M) (the DHO stok solution was prepared in loona OK external solution); 2) extraellular superfusion with solution ontaining 2 M DHO; 3) extraellular superfusion with Na+-free solution. DHO is a speifi and reversible inhibitor of the Na/K pump inxenopus ooytes (Shweigert et al., 1988; LaTona, 199). period of 4-5 min was allowed after DHO addition before data sampling. Only 1-2 min is required to obtain omplete blok. The harge moved (Q) was determined by diret numerial integration of the subtrated urrent reords. Usually, the integration was performed over 35 ms starting 4-8,us after the start of the voltage step. The relaxation rate was determined by fitting the transient omponent of the subtrated urrent reords to a single exponential deay equation as follows: I, = It(O) exp(-kt), where It is transient urrent, t is the time, It(O) is the magnitude of It extrapolated to time, and k is the exponential relaxation rate. The integrals and the relaxation rates of the transient urrents for eah voltage step were alulated using the CLMPN and CLMPFIT software modules of PCLMP. Further analysis, least-square urve fitting, and preparation of figures were done with SIGMPLOT software version 5. (Jandel Sientifi, Corte Madera, C). Curve fit parameters are reported ± SD of the value obtained from the least-squares fitting proedure. Experiments were performed at room temperature (-22 C). E P%^ -1uu Volume 66 Marh 1994 I I I C,. i, I. n) vi 1d ,ll l l 7I7I II II RESULTS Extraellular Na-sensitive transient urrent Fig. 1 shows urrent reords obtained from an ooyte in loona OK external solution and internally perfused with 5 mm Na+, 5 mm TP, 5 mm DP, K+-free solution: onditions that favor the operation of the eletroneutral Na/Na exhange mode of the Na/K pump (Glynn, 1985). The reords show the average urrent eliited by voltage lamp pulses to -16, -12, -8, -4, and +4 mv from a holding potential of -4 mv as diagrammed in Fig. 1. Fig. 1 C shows average urrent reords produed with the same pulse protool but during superfusion with ONa OK solution. The transient omponent of these reords is smaller than the orresponding reords in Fig. 1. Fig. 1 D shows the Na.-sensitive urrents obtained by subtration of urrent reords in the absene of Na+ (C) from those in its presene (). The hyperpolarizing steps resulted in transient inward urrents whih deayed exponentially to zero. Returning to the holding potential produed transient outward urrents with slower relaxation time onstants. The depolarizing steps aused transient outward urrents and the return steps produed transient inward urrents. The absene of steady-state urrent at any voltage tested onfirmed the expetation that there should be no steady-state urrent generated by the eletroneutral Na/Na exhange mode of the Na/K pump. Voltage-dependene of the Nao-sensitive transient urrent The voltage dependene of the Nao-sensitive transient urrent reords from Fig. 1 D is shown in Fig. 2. The quantity of harge moved during the on (Qon, open irles) and off (Qoff, filled irles) of the pulses are shown in Fig. 2. The 3.) Time/ms FIGURE 1 Na-sensitive transient urrents. () Voltage pulse protool. The voltage pulses were done in inrements of 4 mv from -16 to + 4 mv and are 4 ms in duration. Holding potential -4 mv. () Current time ourse in loona OK external solution. (C) Current time ourse in ONa OK external solution. Sodium removal redued the transient urrent both at the on and offof the pulses. (D) Na.-sensitive urrent determined by subtration of reords in the absene of sodium (C) from those in its presene (). The urrent delines to zero in the steady-state. Sodium removal typially produed a D.C. offset in the urrent traes (see C andd). This probably reflets hanges in the liquid juntion potential between the lona OK and ONa OK external solutions and the agar bridges. Sine the eletrophoreti mobilities of tetramethylammonium+ and Na+ are similar, the hange in juntion potential is expeted to be small. The hange in bath voltage was measured on hanging from ONa OK to lona OK solutions and was found to be about 1 mv. Given an ooyte membrane impedane of 5 Kfi of the experimentally ontrolled area, a 1-mV hange injuntion potential would be suffiient to explain the -2 n urrent offset. following equation was fitted to the values of Q.n: Q(V) = Qmin + Qtot/{l+ exp[zq(vq - V)F/RT]}, (1) where Qtot is the total amount of harge moved, zq represents an apparent valene, Vq is the midpoint potential, V is membrane potential, and F, R, and T have their usual meanings. Fig. 2 shows the voltage dependene of the relaxation rate onstants (k) of the transient urrents. The value at the holding potential (-4 mv; filled irle) is the average of the relaxation rate of the Na-sensitive transient urrent measured at the off of the pulses. The relaxation rate (solid line) an be desribed as the sum of a forward voltage-independent rate onstant and a reverse voltage-dependent rate oeffiient

4 Holmgren and Rakowski Na/K Pump Transient Currents 915 FIGURE 2 U) Cl) 6 2 O -8 Voltage dependene of the Na-sensitive transient urrent. () Voltage dependene of the harge moved during the on (QWm; open irles) and off (Qff; filled irles) of the experiment shown in Fig. 1. Filled symbols obsure the open symbols. Qon and Qoff were alulated by numerial integration of reords in Fig. 1 D. The integration started 6 ps after initiating the voltage step, and was terminated at 35 ms. The solid line is a least-square fit of Eq. 1 to Qon. The best-fit parameters are Qmin = -117 ± 6 pc, Q,tt = 2 ± 13 pc, zq =.81 ±.1, and Vq = ± 3.5 mv. () Voltage dependene of the relaxation rates (k). Open irles, alulated from the on transient urrents. Filled irle, average obtained from the off transient urrents. The SEM is smaller than the radius of the irle. The solid line is a least-square fit of Eq. 2 to the data. The best-fit parameters are ak = s-1, Zk =.76 ±.11, and Vk = mv. The dashed lines represent the forward and bakward rate oeffiients from Eq. 2. (dashed lines) aording to Eq. 2: k(v) = ak{l+ exp[zk(vk - V)F/RT]}, (2) where ak is a voltage-independent rate onstant, Zk is the apparent valene, and Vk is the value of voltage in whih k = 2ak. Equations 1 and 2 are derived from a simple twostate model, whih requires that zq = Zk, and that Vq = Vk (Rakowski, 1993). The best-fit values for the parameters are given in the figure legend. Intraellular substrate dependene of harge transloation Fig. 3 shows Nao-sensitive transient urrents obtained under normal onditions, i.e., the ooyte was internally perfused with 5 mm Na+, 5 mm DP and TP, and both the internal and external solutions were nominally K+-free. Removal of intraellular Na+ diminished the Nao-sensitive transient urrents almost ompletely (Fig. 3 ). Fig. 3 E displays the average steady-state harge distribution of two experiments in the presene (open irles) and absene (filled irles) of intraellular Na+, respetively. Solid and dashed lines represent the best fit to the data; parameter values are given in the figure legend. The harge moved in the absene of Na+ is very small making the fit proedure unreliable. Nevertheless, for omparative purposes, we obtained a fit by onstraining zq to be the same as that found under normal onditions. pproximately 86% of the total amount of harge was redued by intraellular perfusion with Na+-free solution. Sine intraellular Na+ is a required substrate for Na/Na exhange, its absene is expeted to stop the exhange. Nao-sensitive transient urrents were absent in an experiment that was started without intraellular nuleotides (Fig. 3 C). Intraellular perfusion of this same ooyte with a solution ontaining 5 mm of TP and 5 mm of DP resulted in the appearane of Na.-sensitive transient urrents (Fig. 3 D). Fig. 3 F shows the steady-state harge distribution of Q.. in the presene (open irles) and absene (filled irles) of intraellular nuleotides, respetively. Solid and dashed lines represent the best fit to the data; parameter values are given in the figure legend. The absene of nuleotides abolished -8% of the harge. lthough hydrolysis of TP is not neessary for Na/Na exhange to our (Garrahan and Glynn, 1967b), the presene of both TP and DP are required (DeWeer, 197; Glynn and Hoffman, 1971; Cavieres and Glynn, 1979). Effet of extraellular addition of oligomyin and Kg Fig. 4 shows Na-sensitive transient urrents under normal onditions with the exeption that the loona OK and the ONa OK external solutions ontained.5% of DMSO (used to dissolve oligomyin ). ddition of 1 p,g ml-' of oligomyin to the extraellular solutions redued the Na.-sensitive transient urrents (Fig. 4 ). Fig. 4 E shows the average steady-state harge distribution under ontrol onditions (open irles) and after the addition of oligomyin (filled irles) from this and one additional experiment. Oligomyin bloked -8% of the harge transloated. In the absene of K+, ouabainsensitive sodium efflux in resealed ghost ells is inhibited 91% by 1 [kg ml-' of oligomyin (Garrahan and Glynn, 1967b). Fig. 4 C shows Na.-sensitive transient urrents under normal onditions. ddition of 5 mm of K+to the external solutions resulted in the abolition of the transient urrents (Fig. 4 D). Fig. 4 F displays the average harge distribution from this and two additional experiments in ontrol onditions (open irles) and after adding external K+ (filled irles). ddition of external K+ is expeted to ativate forward pumping by the Na/K pump, resulting in a redution of the transient urrents and the appearane of steady-state pump urrent (ahinski et al., 1988). veraging the last 15 ms of the traes from Fig. 4 D gives a steady-state urrent versus voltage relationship that saturates at positive potentials at a value of -5 n (data not

5 916 iophysial Journal Volume 66 Marh [- r--iri -I -I ri I 2 I I -2 L- I I I X -2 L I L L I I 15 C 15 D L E F FIGURE 3 Effet of intraellular substrates on harge transloation. () Nao-sensitive transient urrents in response to voltage pulses in inrements of 4 mv from -14 mv to + 2 mv under normal onditions, i.e.: the internal solution ontained 5 mm Na+, 5 mm TP, and 5 mm DP, and was K+-free. () Nao-sensitive transient urrents after 15 min of internal perfusion with a solution in whih sodium sulfamate was replaed by NMG sulfamate. and are from the same ooyte. (C) Na-sensitive transient urrents obtained in the absene of both TP and DP. (D) Na-sensitive transient urrents after 3 min of intraellular perfusion with a solution ontaining 5 mm of TP and 5 mm of DP. C and D are from the same ooyte. ll urrent reords shown in this figure are the average of eight runs of the experimental protool. The data was digitally filtered with a time onstant of.3 ms after averaging. (E) verage steady-state harge distribution from urrent reords of two experiments like the one desribed in Fig. 3, and. Open irles, harge distribution in the presene of intraellular Na+. The solid line is a least-square fit of Eq. 1 to the data. The best-fit parameters are Qmin = ± 5.8 pc, Qtot = 143 ± 15 pc, zq = , and Vq = ± 5.4 mv. Filled irles, harge distribution in the absene of intraellular Na+. The dashed line is a least-square fit of Eq. 1 to the data with the onstraint that zq = The best-fit parameters are Qmin = pc, Qtot = 2.2 ± 1.3 pc, and Vq = mv. (F) Steady-state harge distribution of Qon from urrent reords shown in 3, C and D. Open irles, harge distribution in the presene of intraellular nuleotides. The solid line is a least-square fit of Eq. 1 to Qon. The best-fit parameters are Qmin = pc, Qtot = pc, zq = 1.14 ±.8 and Vq = mv. Filled irles, harge distribution in the absene of intraellular Na+. The dashed line is a least-square fit of Eq. 1 to Qon in the absene of nuleotides with the onstraint that zq = The best-fit parameters are Qmin = pc, Qtot = pc, and Vq = mv. shown). etween 3 and 4 n of maximal Na/K pump urrent per ooyte has been previously reported under similar ioni onditions (Rakowski et al., 1991). Sine the area of membrane from whih we were reording was -1/5 of the total area of an ooyte, we should expet steady-state Na/K pump urrents of the order of 6-8 n, whih is lose to the value obtained experimentally. The transient omponent of the Na/K pump urrent was diminished by approximately 88% after ativation of forward pumping. Comparison between Nao-sensitive and DHO-sensitive transient urrents Nao-sensitive and DHO-sensitive transient urrents, measured in the same ooyte, are shown in Figs. 5, and, respetively. t the beginni'ng of the experiment, the ell was superfused with loona OK solution and 2 repetitions of a pulse protool onsisting of voltage lamp pulses, every 2 mv, from -16 to mv from a holding potential of -4 mv, was applied. Subsequently, the ell was superfused with ONa

6 Holmgren and Rakowski Na/K Pump Transient Currents C 4 D C) Q U) L- a -C E T F FIGURE 4 Inhibitory effet of oligomyin and external K+ on pre-steady-state harge transloation. () Na-sensitive transient urrents obtained under normal onditions, but the external solutions ontained.5% of DMSO. The pulse protool was the same as desribed in Fig. 3. () Na-sensitive transient urrents obtained in the presene of 1,g ml-' of oligomyin in the extraellular solutions. and are from the same ooyte. (C) Normal Na-sensitive transient urrents in response to the same pulse protool as desribed in Fig. 3. (D) DHO-sensitive transient urrents in the presene of 5 mm of K+ in the external solution. C and D are from the same ooyte. (E) verage steady-state harge distribution from urrent reords of two experiments like the one desribed in Fig. 4, and. Open irles, harge distribution under ontrol onditions. The solid line is a least-square fit of Eq. 1 to the data. The best-fit parameters are Qmin = pc, Qtot = 67.4 ± 5.9 pc, zq = , and Vq = ± 4.5 mv. Filled irles, harge distribution in the presene of 1,g ml- of oligomyin in the external solutions. The dashed line is a least-square fit of Eq. 1 to the data with the onstraint that zq =.91. The best-fit parameters are Qmin = pc, Qtot = 13.8 ± 6.7 pc, and Vq = mv. (F) verage steady-state harge distribution from urrent reords of three experiments like the one desribed in Fig. 4, C and D. Open irles, harge distribution in normal onditions. The solid line is a least-square fit of Eq. 1 to Qon. The best-fit parameters are Qm1n = pc, Qtot ± = pc, zq = 1.19 ±.15, and Vq = mv. Filled irles, harge distribution in the presene of 5 mm of K+ in the external solutions. The dashed line is a least-square fit of Eq. 1 to the data with the onstraint that zq = The best-fit parameters are Qmin = -14. ± 3.6 pc, Qtot = 28.6 ± 6.3 pc, and Vq = -44. ± 14.4 mv. OK solution and 2 more repetitions of the same pulse protool were done. The ell was returned to loona OK in order to reativate the Na/Na exhange mode of the Na/K pump. fter 2 additional repetitions of the protool were performed in this solution, the ooyte was superfused with the same external solution but now ontaining 2,uM DHO and the protool was repeated for a final 2 times. Transient urrent reords were obtained by subtration of average urrent reords in loona OK external solution from: 1) those obtained in the absene of extraellular Na (Fig. 5 ), and 2) those obtained after the addition of 2,uM DHO (Fig. 5 ). Fig. 5 C shows the harge movement assoiated with the Na-sensitive and DHO-sensitive urrent. Solid and dashed lines represent the best fit to Q.,, alulated from the Na- sensitive and DHO-sensitive transient urrents, respetively. The total amount of harge moved as measured from the DHO-sensitive transient urrent was approximately 2% larger than that determined from the Na.-sensitive urrent. This differene is not unreasonable. t the high onentration of DHO used in this experiment (2,uM) we expet a 98% blok of the Na/K pump given a Ki of.4,um for inhibition of the Na/K pump by DHO in Xenopus ooytes

7 918 1 iophysial Journal 1 I IFIrI ri Volume 66 Marh ' -2,.- -4 L C E -Q N -o a z D F I, 3 1( r) I -\ % %%.~~~~~~~ FIGURE 5 Comparison of Na-sensitive and DHO-sensitive transient urrents. The pulse protool was similar to that in Fig. 1 exept that the range of voltages explored was from -16 to mv. () Na-sensitive transient urrents obtained by subtration of urrent reords in the ONa OK solution from those obtained in 1ONa OK solution. () DHO-sensitive transient urrents obtained by subtration of urrent reords in the presene of DHO from those in its absene. The reords in and were obtained from the same ooyte. The Na-sensitive transient urrents are quite similar to the DHO-sensitive transient urrents. (C) Voltage dependene of Q. Qon and Qff for the Na-sensitive transient urrents are given by open and filled irles, respetively. The solid line is a least square fit of Eq. 1 to the data. The best fit parameters are Qmi,n = pc, Qtot = pc, zq = 1.1.2, and Vq = -88 ± 4 mv. Qon and Qff for the DHO-sensitive transient urrents are given by open and filled triangles, respetively. The dashed line is a least square fit of Eq. 1 to the data. The best fit parameters are Qmin = -381 ± 2 pc, Qtot = 46 ± 26 pc, zq = 1.7 ±.13, and Vq = mv. (D) Normalized harge at the on of the pulses versus membrane potential. Open irles, Na-sensitive transient data; open triangles, DHO-sensitive transient data. The solid lines through these data represent the best fit to the equation: {Q(V) Qmin}/Qtot = 1/{1 + exp[zq(vq - V)F/RT]}. (E) Voltage dependene of k for the Nao-sensitive transient urrents. Open irles, values of k from the on of the pulse. Filled irle, average k of the transient urrents from the off. The SEM is smaller than the radius of the irle. The solid line is the best fit of Eq. 2 to the data. The best fit parameters are ak = 186 ± 4 s-1, Zk = , and Vk = -147 ± 1.5 mv. The dashed lines represent the forward and bakward rate oeffiients from Eq. 2. (F) Voltage dependene of k for the DHO-sensitive transient urrents. Open irles, values of k from the on of the pulse. Filled irle, average k of the transient urrents from the off. The SEM is smaller than the radius of the irle. The solid line is the best fit of Eq. 2 to the data. The best fit parameters are ak = 174 ± 11 s-5, Zk =.82 ±.2, and Vk = mv. The dashed lines represent the forward and bakward rate oeffiients from Eq. 2. Measurements of the relaxation rate of urrent reords at positive potentials are subjet to large errors beause the urrents are small and the time onstant long. For these reasons the value of k at mv in E and the value of k at -2 mv in F were not inluded in the urve fitting proedure. (Shweigert et al., 1988). On the other hand some sodium from Nao-sensitive (n = 6, and C) and DHO-sensitive probably remained in the hamber after a hange to Na-free (n = 5, and D) transient urrents. solution due to inomplete Na removal. Normalized values Table 1 summarizes the mean ± SE of the parameters of Qon from Nao- and DHO-sensitive transient urrents are obtained from the data from individual ooytes to Eqs. 1 and shown in Fig. 5 D. Fig. 5, E and F, show the voltage de- 2. The parameters obtained from Nao-sensitive and DHOpendene of k from the Nao- and DHO-sensitive transient sensitive transient urrents were not statistially signifiantly urrents, respetively. Fig. 6 is a summary of the voltage different (right-most olumn). Therefore, it is likely that both dependene of the harge moved and the relaxation rates signals report the same event. single fator analysis of

8 Holmgren and Rakowski Na/K Pump Transient Currents 919 FIGURE 6 Summary of the voltage dependene of pre-steady-state harge transloation. () Voltage dependene of the normalized harge from Na-sensitive transient urrents (n = 6). () Membrane potential dependene of the normalized harge from DHO-sensitive transient urrents (n = 5). Open irles in and are the normalized harge moved at the on and filled irles represent the normalized harge moved at the off of the pulses. Solid lines are the best fit to the data using the normalized version of Eq. 1. The best fit parameters are: ()zq =.87 ±.5, Vq = ± 1.7 mv; () zq =.86 ±.5, Vq = mv. (C) k from Naosensitive urrents and (D) k from DHO-sensitive urrents. Solid lines are the result of the best fit to the data using Eq. 2. The best fit parameters are: (C) ak = 272 ± 34 s-,zk =.62 ±.17, and Vk= -132 ± 13 mv; (D) ak = 249 ± 39 s-1, zk =.69.29, and Vk = mv. oltzmann fits of averaged data tend to give an understimate of the exponential steepness fator Zk. The mean values obtained from the best fit parameters of the individual experiments are shown in Table 1. The dashed lines in C and D represent the forward and bakward rate oeffiients from Eq. 2. a.n ) E z C D TLE 1 Comparison of harge movement parameters from Nao-sensitive and DHO-sensitive experiments Nao-sensitive DHO-sensitive Parameter (n = 6) (n = 5) t test* Zq-on.96 ± ±.7.1 < p <.2 Zq-off.95 ±.5.85 ±.6.2 <p <.5 Qtot (pc) 23 ± ± 34.2 <p <.5 Vq (mv) ± ± < p <.5 ak (S-') 266 ± ± 31.2 <p <.5 Zk.8 ±.5.86 ±.1 p >.5 Vk (mv) ± 9. p >.5 * two-tailed t test was performed for eah parameter. t a signifiane level of.5, all the omparable means between the Nao-sensitive and DHOsensitive transient urrents are not statistially signifiantly different. variane test was performed on zq(on), zq(off) and Zk for Nao-sensitive transient urrent with the result that the mean of these three parameters were not statistially different (.5 <p <.1). Similar results were obtained when the test was performed for DHO-sensitive transient urrents. Comparison of Na/K pump density Qon and Ooff and alulation of Fig. 7 shows Qon vs. Qoff measurements for pulses between -16 and + 6 mv for 16 experiments in whih the Na/K pump was stopped by any of the three different experimental methods. Within experimental error, equality of Q.n and Qoff is found for all voltages explored. The mean value of the total amount of harge moved per ooyte for the DHO-sensitive transient urrents was 27 ± 3 pc. Given a linear apaity L 1 C Qon/pC FIGURE 7 Equality of on and off harge movement. Values of Qon versus Qoff obtained from pulses between -16 and +6 mv are plotted. Results were obtained from 16 experiments in whih Na/Na exhange was stopped by any of the three methods desribed under Materials and Methods. Open irles, experiments in whih the intraellular [Na] was 5 mm (12 experiments). Open triangles, experiments in whih the intraellular [Na] was 25 mm (four experiments). The solid line is the theoretial relationship QDn = Qoff of.18,uf per ooyte (Vasilets et al., 199), a speifi membrane apaitane of 1 tkf/m2, and further assuming that one net harge is moved per pump site and that the area of membrane under experimental ontrol is -2% of the total membrane area, we find that the Na/K pump site density in the vegetal hemisphere is 46 ± 6,um2.

9 92 iophysial Joumal Volume 66 Marh 1994 bsene of rising phase in the transient urrents Fig. 8 shows DHO-sensitive transient urrents obtained with the on of voltage pulses of 1 ms in duration from -4 mv to ommand potentials of -1, -8, -6, -2,, and 2 mv. Fig. 8 displays the same urrent reords expanded in both the x and y diretions, starting the x axis at the time of the onset of the voltage steps, i.e., 1.68 ms. rising phase was not observed even in transient urrents resolved from -5,us after the beginning of the voltage step. fast urrent omponent was not temporally resolved (see Disussion). DISCUSSION Charge transloation and Na/Na exhange. Five modes of operation of the Na/K pump are well established (Glynn, 1985): 1) Forward and reverse TP-driven Na/K exhange, 2) K/K exhange, 3) TP-driven Na-efflux, 4) TP-driven Na/Na exhange, and 5) Na/Na exhange without net hydrolysis of TP. The absene of K+ in both the internal and external solutions should prevent both modes 1 and 2 of pump operation. Under the onditions used to obtain Nao-sensitive transient urrents, i.e., absene of extraellular Na+ and K+, the Na/K pump an atalyze an TP-driven Na+ extrusion (Garrahan and Glynn, 1967a; N `N FIGURE 8 bsene of rising phase in DHO-sensitive transient urrents. () DHO-sensitive urrents obtained from a holding potential of -4 mv to omand potentials of -1, -8, -6, -2,, and 2 mv. The pulses were 1 ms in duration, and the data was sample every 3,us. () Same urrent traes as shown in but expanded in both x and y axes. The x axis was displaed in order to start at the onset of the pulse. Glynn, 1985). This mode ours by normal Na+ efflux, followed by a slow hydrolysis of the phosphoenzyme. The stoihiometry and the turnover rate have been estimated to be -3 Na+/1TP and 3 s-5, respetively (Cornelius, 1989). With 46 pumps pm-2 and a total area of -3.6 X 16 plm2 (area of the dome under experimental ontrol) we expet a steadystate urrent mediated by TP-driven unoupled sodium efflux between 1.8 and 3.6 n. The possible presene of unoupted sodium efflux of this magnitude should not produe a signifiant differene between Na- and DHO-sensitive transient urrents. In most of the experiments, we did not observe steadystate urrent in either the Nao- or DHO-sensitive urrents (e.g., Figs. 1, 3, 4, and 5). In the absene of K+ and DP, but the presene of high Na+ onentrations, the pump is known to operate in a TP-driven eletrogeni Na/Na exhange mode (probably 3 Na/2 Na) (Lee and lostein, 198). Sine the ooytes were internally perfused with 5 mm of DP, we an exlude this mode of operation as a possible ontaminant of our results. The onditions of these experiments, i.e., the absene of K+in the internal and external solutions, the presene of intra- and extraellular Na+, and the presene of intraellular TP and DP, strongly favor the operation of the Na/K pump in its lassial 3 Na/3 Na exhange mode without net hydrolysis oftp (Glynn, 1985) (Fig. 9). s expeted for this mode ofpump operation, harge transloation is abolished by the lak of TP and DP (Fig. 3), intraellular (Fig. 3) or extraellular Na+ (Fig. 1), and is bloked by DHO (Fig. 5), oligomyin (Fig. 4) and the ativation of forward Na/K pumping (Fig. 4). We onlude, therefore, that the harge transloation studied here originates from the Na/K pump operating in its eletroneutral Na/Na exhange mode. Voltage dependene of the transient urrent relaxation rate. Charge transloation was analyzed by a simple model in whih the harge is distributed between two states and only the bakward rate oeffiient is voltage-dependent DP Na3 E1 TP (Na3)E1-P P-E2 Na3 MIa+^t ~Na+e 3 E1lTP P-E2 FIGURE 9 Simplified kineti sheme of Na/Na exhange. In the absene of K+ and the presene of DP, TP, and Na+, the Na/K pump operates mainly as a three for three Na/Na exhange. The enzyme an assume two major onformational states designated E1 and E2. E1 has ion-binding sites faing the ytoplasm and E2 has ion-binding sites faing the extraellular medium. When the pump is phosphorylated by TP in the E1 state, Na+ ions beome oluded (i.e., trapped inside the protein). fter the onformational hange to E2, Na+ ions are release to the extraellular medium. ll reations are reversible under the onditions required for Na/Na exhange. (Figure modified from L3uger, 1991, p. 178.)

10 Holmgren and Rakowski Na/K Pump Transient Currents 921 (Rakowski, 1993). This asymmetrial voltage dependene is expeted for aess hannel models of external Na+ binding (at least at low levels of aess hannel oupany) (Gadsby et al., 1993). The data shown in Figs. 2, 5, and 6 support this aess hannel model in that the relaxation rate appears to beome voltage-insensitive as the membrane is depolarized. dequay of a pseudo-two state model The harge distribution data reported by Nakao and Gadsby (1986) obey a oltzmann distribution in whih approximately a single positive harge is transloated aross the entire membrane field. On the other hand, as noted by De Weer (199), the voltage dependene of k behaves as if the harge traverses only 4% of the entire membrane field. Similarly, Rakowski (1993) found that the apparent valene alulated from the harge distribution (zq) is lose to 1, whereas the apparent valene derived from the urrent relaxation rate (Zk) was -.5. We find that zq and Zk are not signifiantly different either for Nao- or DHO-sensitive transient urrents (see Table 1). The presene of a rising phase in the transient urrents would be strong evidene that the harge transloation ours between more than two states. In Xenopus ooytes, previous studies of harge movement assoiated with the Na/Na exhange were done with a two-miroeletrode voltage lamp (Rakowski, 1993). With this tehnique, the time onstants of the voltage steps are usually between 1 and 1.5 ms, limiting the study to events that have relaxation rates slower than about 2 ms. The open-ooyte vaseline-gap tehnique has allowed us to make voltage steps with time onstants as fast as 2,is. The apaity transient assoiated with these steps ended at most in 6 p,s. No evidene of a rising phase has been observed for times longer than this temporal resolution (Fig. 8). The fast omponent of the DHO-sensitive transient urrent deteted in Fig. 8 ould reflet equilibration of ions within an external aess hannel, however, this urrent omponent was not temporally resolved. There is one major inonsisteny between our data and the preditions of a simple pseudo two-state model. The value of Vq, whih orresponds to the voltage at whih the forward and bakward rate oeffiients are equal, should be the same as Vk. This disrepany indiates that the two-state model is oversimplified. Our values of Vk (--13 mv) are usually muh more negative than the orresponding values of Vq (-6 mv). With this exeption a simple two-state model desribes the pre-steady-state urrent reasonably well between membrane potentials of -14 and +6 mv. Multistate models predit that k should beome a saturating sigmoid funtion of membrane potential. In order to expand the experimentally aessible range of voltage, solutions ontaining niflumi aid (3,uM) to blok hloride hannels (Kubo et al., 1993) and gadolinium (1,uM) to blok strethativated ation hannels (Yang and Sahs, 1989) are presently being used. Hilgemann (personal ommuniation), using a giant path tehnique, has reported that k saturates at extreme negative voltages. Comparison of Nao-sensitive and DHO-sensitive transient urrents Rakowski (1993) found no differene between Na-sensitive and DHO-sensitive transient urrents. Our results support those findings (see Table 1). This similarity is not unexpeted sine both subtrations should have similar harateristis if the same harge transloating steps have been isolated by the experimental design. Comparison of parameter values with previous measurements t an extraellular [Na+] of 9 mm, Rakowski (1993) found that zq was lose to 1, Vq was -4 mv, Zk was -.5, ak was -1 s-5, and Vk was -5 mv. We found that zq and zk were both lose to 1, Vq was --6 mv, ak was -25 s-1, and Vk was -13 mv. The fat that we performed our experiments in the nominal absene of internal and external K+ may have ontributed to have faster ak. Rakowski (1993) did not have aess to the intraellular medium, so his experiments were done in the presene of intraellular K+. Intraellular K+ may displae some of the enzyme from the E1lTP state to preeding potassium-assoiated states. If those states relax to the ElTP state slowly, then the value of the overall harge transloation rate would be slower in the presene of internal K+. Our values of ak are loser to those estimated from guinea pig ventriular myoytes (Nakao and Gadsby, 1986) but, those experiments were performed at 37C. The value of Vq we obtained is -2 mv and -4 mv more negative than that reported by Rakowski (1993) in Xenopus ooytes and Nakao and Gadsby (1986) in ardia myoytes, respetively. Sine mid-point voltages may be affeted by suh fators as surfae harge density, it is probably not worthwhile to speulate about differenes that may our between speies. However, it may be worthwhile to ompare data obtained from Xenopus ooytes further. The Q vs. V relationship is known to shift toward positive potentials with inreasing [Na]o (Rakowski, 1993). If the Q vs. V relationship is similarly affeted by internal [Na], this may explain the quantitative differene between the results. Our values of Vk are also more negative than the values obtained by Rakowski (1993). Comparison of Qon and Qoff and alulation of Na/K pump density Within experimental error, equality Of Q.n and Qoff was found for all voltages explored. This is in good agreement with our results in Xenopus ooytes using the twomiroeletrode voltage lamp tehnique (Holmgren and Rakowski, 1993a). pproximate equality of Qon and Qoff was found for all voltages and was independent of holding potential in the range -1 to mv. The previous disrepanies between Qon and Qoff (Rakowski, 1993) were eliminated when the urrent integral was alulated by diret integration rather than the extrapolation method originally used.

11 922 iophysial Journal Volume 66 Marh 1994 The Na/K pump site density in the vegetal hemisphere was found in Results to be 46 ± 6 gum-. If Xenopus ooytes have -2/3 of their Na/K pumps in the vegetal pole (W. Shwarz, personal ommuniation) and assuming that the area of the vegetal and animal poles are equal, our estimation of pump density orresponds to -34 pumps,um-2 if the pumps were distributed homogeneously over the ell surfae. This result is in good agreement with values of pumps pum-2 obtained from measurements of transient urrents of the whole ooyte with the two-miroeletrode voltage lamp tehnique (Rakowski, 1993) and values of ouabain binding sites,um-2 (Vasilets et al., 1991). Substrate dependene and origin of the harge transloation The harge transloation studied here appears to result from the funtioning of the Na/K pump in its eletroneutral Na/Na exhange mode, sine it depends on the presene of intraellular Na+ and nuleotides, and extraellular Na+. Oligomyin is known to blok the spontaneous hange from (Na)3E,-P to P-E2 Na3 (Glynn, 1993) (see Fig. 9). Oligomyin also bloked Na-sensitive transient urrents, suggesting that harge transloation requires that the deolusion/olusion reation our. Nakao and Gadsby (1986) found that harge transloation in myoytes an our in the nominal absene of internal DP, whih would fore the pump to operate in the right-hand half of Fig. 9. Measurement of oligomyin -sensitive transient urrents ould be a useful experimental tool. If oligomyin- sensitive transient urrents have the same harateristis as Nao- and DHO-sensitive transient urrents, this would onfirm the suggestion of Nakao and Gadsby (1986) that only the deolusion/olusion reation makes a kineti ontribution to the urrent relaxation rate. We are indebted to Drs. David Gadsby and Paul De Weer for helpful advie and ritiism. We thank Dr. F. ezanilla for tehnial assistane with the open-ooyte vaseline-gap tehnique. Supported by National Institutes of Health Grant NS REFERENCES ahinski,., M. Nakao, and D. C. Gasby Potassium transloation by the Na+/K+ pump is voltage insensitive. Pro. Natl. ad Si. US. 85: Cavieres, J. D., and I. M. Glynn Sodium-sodium exhange through the sodium pump: the roles of TP and DP. J. Physio. (Lond.). 297: Chandler, W. K., R. F. Rakowski, and M. F. Shneider nonlinear voltage-dependent harge movement in skeletal musle. J. Physiol. 254: Cornelius, F Unoupled Na+-efflux on reonstituted shark Na, K-TPase is eletrogeni. iohem. iophys. Res. Commun. 16: DeWeer, P Effets of intraellular 5'DP and orthophosphate on the sensitivity of sodium efflux from squid axon to external sodium and potassium. J. Gen. Physiol. 56: DeWeer, P The Na/K pump: a urrent generating enzyme. In Regulation of Potassium Transport aross iologial Membranes. L. Reuss, G. Szabo, J. M. Russell, L. Reuss, G. Szabo, and J. M. Russells, editors. University of Texas Press, ustin, TX Gadsby, D. C., R. F. 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