Charge Translocation of H,K-ATPase and Na,K-ATPase M. STENGELIN," A. EISENRAUCH," K. FENDLER," G. NAGEL," H. T. W. VAN DER HIJDEN! J. J. H. H. M. DE PONT! E. GRELL, AND E. BAMBERff aMax-Planck-lnstitutfur Biophysik W-6000Frankfurt, Germany bDepartment of Biochemistry UniversityofNijmejen 6500 HB Nijmejen, The Netherlands The H,K-ATPase and Na,K-ATPase belong to the class of P-type ATPases and have a homology in primary structure of more than 60%. Their transport mechanism can roughly be described by a similar scheme (Albers-Post cycle). The major difference is that the H,K-ATPase is an electroneutrally operating pump, whereas the Na,KATPase transports a net charge. An important step toward a better understanding of the transport mechanism of ion pumps is to measure the charge translocation directly. Recently the use of artificial planar membrane techniques has proved to be very suitable in studying the electrogenic properties of ion pumps. It is possible to incorporate ATPases into a black lipid membrane (BLM) or to adsorb ATPase-containing membranes to a BLM.'" In the latter case the systems are capacitively coupled. The advantage of the first method is the free accessibility of both sides of the membrane protein. In particular, a defined voltage can be applied. The signal-to-noise ratio, however, is so far two orders of magnitude better in the case of the latter method. Therefore, steady-state current voltage measurements were performed with incorporated protein and transient kinetic measurements with adsorbed membranes. An electrical signal can be induced by an ATP concentration jump via photolysis of caged ATP, a photolabile-protected inactive derivative of ATP.' These currents were measured under a variety of conditions, allowing analysis of the electrical signal in terms of relaxation times associated with different partial reactions of the enzymatic cycle. Assignment of the relaxation times to partial reactions was made, and rate constants for some of the reactions were given. In particular, the electrogenic steps were localized. In this paper we first discuss transient currents generated by H,K-ATPasecontaining vesicles adsorbed to a lipid bilayer. Then we focus on the Na,K-ATPase. As the Na,K-ATPase generates a stationary current, not only the transient signals but also the steady-state currents were analyzed. MATERIALS AND METHODS Transient Currents Generated by ATPase-Containing Membranes Adsorbed to a BLM
H,K-ATPase from pig stomach and Na,K-ATPase from pig kidney were prepared as previously des~ribed.83~ ATPase-containing vesicles or membrane fragments 170
STENGELIN et al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
171
were adsorbed to a BLM (FIG. 1) as described elsewhere.'-',"' A rapid ATP concentration jump was generated by photolysis of caged ATP. The released ATP activates the ATPase and a transient current can be measured. Steady-State Current Voltage Measurements with Incorporated Na,K-ATPuse Incorporation of the Na,K-ATPase into planar lipid membranes was performed by the method of Montal and Muellerll with minor modifications, as described
rq
A
FlGURE 1. Schematic representation of the bilayer setup. (A) Teflon cell with black lipid membrane and adsorbed H,KATPase vesicles. (B) Proposed arrangement of vesicles and underlying lipid membrane. (C) Equivalent circuit diagram of the two membranes in series. G,,,represents the ohmic conductance of the combined parts of the black lipid membrane and adsorbed ATPase vesicles. G,, is the conductance of the residual vesicle membrane. C,, and C,, are the corresponding capacitances. I, designates the pump current generator.
7,K-ATPase
B
II ELM caged ATP
C
previously.I2-l4Monolayers from a solution of 2% glycerolmonooleate in hexadecane and from some microliters of a suspension of ATPase-containing membrane fragments were formed on the surface of electrolyte reservoirs in the two halves of a teflon cuvette. The electrolyte levels were raised until the monolayers fold to form a bilayer over the hole in a septum (diameter 150-200 pm), dividing the cuvette. Bilayer formation was monitored by controlling membrane conductance and capaci-
172
ANNALS NEW YORK ACADEMY OF SCIENCES
tance. The ATPase was activated by an ATP concentration jump after UV illumination of caged ATP. Current-voltage curves were obtained by applying various voltages using a constant voltage source. As every IV curve summarizes the results of several experiments with different current amplitudes, data are expressed as normalized current amplitudes I/bversus applied voltage. The currents were converted and amplified by a current-voltage converter and stored on a digital storage oscilloscope.
ELECTRIC SIGNALS GENERATED BY THE H,K-ATPase As the H,K-ATPase is an electroneutral pump, no stationary current can be observed. By separation of the H+-transporting half cycle from the K+-transporting part, however, charge translocation was observed during the El --* E2Ptransition. Adding the H,K-ATPase-containing membranes to one side of the cuvette and stirring for about 30 minutes led to adsorption of the membranes to the lipid bilayer. Release of ATP from caged ATP with an UV laser flash generated a biphasic electric signal (FIG.2a). After the addition of SCH 28080 (FIG.2b) or omeprazole (data not shown), specific inhibitors of the H,K-ATF'ase, the signal disappeared. The first phase of the signal represents the movement of a positive charge towards the BLM. The biphasic shape of the signal can be attributed to the electrical properties of the compound membrane. Asum of three exponentials ( T ; ~= 400~-~, 7i1 = lOOs-I, 73' = 10s-1) could be fitted to the transient signal. The model with three exponential components gave
a
d FIGUREZ. Inhibitionof the electric signal by SCH 28080, a specific inhibitor of the H,K-ATPase. The electrolyte contained imidazole buffer 50 mM,pH 6.14, MgClz 3 mM,EGTA 0.25 mM,DTT 0.5 mM, caged ATP 0.146 mM, and 7 = 23%: (a) without SCH 28080; and (b) after the addition of 10 FM S C H 28080.
b
+
10 pM SCH28080
20 PA
i 1
I 0-3
lo-*
Time [s]
lo-'
1oo
STENGELIN el a/.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
G = 0.26 pS/cm
173
'r
FIGURE 3. Conduction dependence of the electric signal. The electrolyte contained imidazole buffer 50 mM, pH 6.2, MgC12 3 rnM, EGTA 0.25 m M ,DTT 0.5 mM, KC1 0.5 mM, glucose 50 pM, hexokinase 1 p, caged ATP 0.26 rnM, 7 = 14%. and T = 300 K. Conductance was increased by the addition of monensin and 1799.
0.001
0.01
0.1
1
Time [ s ]
good fits and was generally used in the part of this study dealing with the H,KATPase. The conductance of the membranes was increased by adding ionophores. As in previous work,' the protonophore 1799 plus the Na+,K+/H+-exchangingcarrier monensin was used. With increasing conductance, the magnitude of the second phase of the electrical signal decreases, and at conductivities of about 3 pS/cm2 the signal becomes monophasic (FIG. 3). In contrast to that with the (Na,K)-ATPase' and the CaATPase,' no stationary current occurs either with or without K+, indicating that the pump operates electroneutrally under stationary conditions. Thus, there must be at least two oppositely directed electrogenic steps.h The basis for the following discussion is the reaction cycle shown in FIGURE4. Effect of Potassium on E2 ++ El Preequilibrium Causes a Reduction in the Electric Signal
The ion selectivity of the electric signal with respect to monovalent cations was investigated.6 Sodium ions had no effect on the signal in the range of 0-10 mM. K+ and Rb+ decreased the peak current with a of 3 mM. The effect was even more pronounced for TI+(FIG.5). With increasing potassium concentrations the magnitude of the electric signal decreased. The signal form, however, did not change (FIG.6). This can be explained if the transition from E2 to El is In this case, enzyme molecules starting in E2
174
ANNALS NEW YORK ACADEMY OF SCIENCES
ElcgATP
11
72
A
L
-e
-
73
Jm
+e
H+I
+
i
-K
1
FIGURE 4. Reaction scheme for the H,K-ATF'ase. before an ATP concentration jump produce only a minimal electric signal, because a slow step precedes the following faster reactions. Potassium shifts the E2 e El preequilibrium towards &,15J6 thus increasing the pool of enzymes that do not produce an electric signal. 72Reflects
the ATP Binding
The ATP concentration was varied in two ways: first, the caged ATP concentration was increased at a constant ATP/caged ATP conversion ratio q; second, -q was changed at a constant caged ATP concentration of 1.3 mM (FIG. 7). The peak current, Ipeok, increased with increasing ATP concentrations. Ipak was higher for identical ATP concentrations if the caged ATP concentration was lower, indicating that caged ATP inhibits the pump. T;' was independent of the ATP and
I max InA/cm21 0
Nd
0
K*
o
Rb* V
TI*
A
\ I
0
3
G
9
12
ImMl FIGURE 5. Dependence of the peak current on monovalent cations. The electrolyte contained imidazole buffer 50 mM, pH 6.0, MgC12 3 mM, caged ATP 0.1 mM, q = 30%, and T = 21°C.
STENGELIN et a/.:CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
175
caged ATP concentration. 7;' became faster for increasing ATP concentrations. For identical ATP concentrations, 7;' was slower if the caged ATP concentration is higher. T~ increased with increasing ATP concentration without a saturation up to 0.3 m M ATP. A model that takes into account the competitive binding of caged ATP to El can explain the ATP depcndcnce of 721n.37.18: k;
-
El caged ATP 7 E l k,
k- is
k:
-f
EIATP + E I P
the caged ATP dissociation rate constant. no K
+
I
c
k: and k,' are the quasi
1 m M K+
I
I
0.00 1
J
0.01
0.1
1
Time [s] FIGURE 6. Electrical signal at various K+ concentrations. The electrolyte contained imidazole buffer 50 mM, pH 6.08, MgClz 3 mM, EGTA 0.25 m M ,DTT 0.5 mM, caged ATP 0.19 mM. -q = 22%. and T = 300 K.
first-order binding-rate constants for caged ATP and ATP, respectively. They depend on the second-order rate constants k,f and k,+: k:
= k:qc,0
(1)
k,' k:(l - q)c; (2) c:!is the caged ATP concentration before the laser flash. The relevant reciprocal relaxation time is a hyperbolic function of the caged ATP
176
ANNALS NEW YORK ACADEMY OF SCIENCES
concentration, if q is held constant:
with (4) 0 0
r,-'
a
0 . 0
600 400
[s-'1
200 0
-1
72
0
t
FIGURE7. ATP dependence of the
300
'A
200
[s-l]
100
0 -1
r3
P I
A
I
A
p
A
2
a A , "
A&nv C
20
10 0
'max
A
1000
gPooo I
,
on+,
electrical signal. The electrolyte contained imidazole buffer 50 mM, pH 6.06, MgC12 3 mM, EGTA 0.25mM, D'IT 0.5 mM,T = 300 K. The ATP concentration was varied in the following way: the conversion rate of caged ATP to ATP was kept at 21% and the caged ATP concentration varied (openfigures).Then the caged ATP concentration was kept constant at 1.3 mM and the conversion rate varied by changing the laser intensity (filled figures). (a-c) The resulting time constants of a fit with three exponentials; (d) peak current.
500
For caged ATP concentrations that are large compared to h.5 ':A of (1 - q)/q:
is a linear function
As FIGURE 8 shows, this model is in accordance with the ATP/caged ATP dependence of 7 2 .
STENGELIN ef al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
FIGURE 8. Fit of the caged ATPATP dependence to the model described in the discussion. (a) 7;' shows a hyperbolic dependence with respect to the free ATP concentration. Fit parameters: b . 5 = 26.1 +M, k,,, = 256 s-I. (b) 72 is a linear function of (1 - q)/q with gradient 0.46 ms and intercept 1.86 ms.
177
b
I
Equations 1 to 6 can be used to estimate the kinetic constants k,+,k:, and k ; . TABLE1 summarizes the results of four measurements under similar conditions. Meanvalueswereky = 3 8 0 s - ' , k : = 1.7.10bs-'M-',andk,+ = 8.3. 1Obs-'M-l. rl Corresponds to the Egi TP
-+
EIP Transition
So far we have attributed 7;' to the ATP binding. In a linear reaction scheme, must be assigned to a step between the formation of ElATP and the electrogenic step or to the electrogenic step itself. TABLE2 summarizes rate constants for the EIATP E I P reaction taken from the literature at different pHs, corrected for T = 300 K. These rate constants are in a time range we can detect and should appear in our signal. A comparison of TABLE2 and the pH dependence of implies that 7;' represents the phosphorylation EIATP + EIP. FIGURE 9 shows the pH dependence of the electric signal. The pH was varied by titrating the electrolyte with HCI. The pH dependence of 7' is bell shaped with a maximum at about pH 5.8. 72 increased monotonously between pH 5 and pH 6.5. 7;'
-+
TABLE1. Kinetic Constants for ATP and Caged ATP Binding to El: Results of Different Experiments under Similar Conditions (Imidazole 50 mM, MgClz3 mM, DTT 0.5 mM, EGTA 0.25 mM. T = 300 K. DH 6.06-6.12)
HK28NO HK0341 HK0841 HK2 I N1
450 540 280 244
2.57 2.48 1.10 0.72
Mean value
379
1.72
13.6 10.0 4.9 4.7 8.29
ANNALS NEW YORK ACADEMY OF SCIENCES
178
TABLE2. Rate Constants for the El .ATP +. E I P Transition Corrected for T = 300 K 5 Wallmark34 L j u n g ~ t r o m ~ ~ 215 S K I
6
I
260 s-l
120 s-'
8
9
43 s-l
6.3 s-l
1.4 150s-1
Time constants 7;' and 7;' that are separated by a factor of about 4 at pH 5.8 show an opposite pH dependence and cannot be distinguished at about pH 6.5-7. 7;' increased with increasing pH. T~ Represents the Electrogenic ErP -+ E2P
Transition
In contrast to the interpretation of similar measurements with the NaK73 is probably not the system time constant but it describes a process of the enzymatic reaction cycle.10
FIGURE 9. pH dependence of the electrical signal. The electrolyte contained imidazole buffer 50 mM, MgC12 3 mM,EGTA 0.25 mM, DTf 0.5 mM, caged ATP 0.26 mM, = 27% T = 300 K. The solution was titrated with HCI. This figure summarizes two subsequent experiments in different pH ranges: (a) Two fast time constants for both experiments; (b) slow time constant 7;'; and (c) peak current.
STENGELIN et al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
179
If the reaction cycle for the H,K-ATPase is similar to the Albers-Post reaction scheme, T~ must be related to a process either preceding the formation of El or following the formation of ElP. The potassium dependence of the electric signal allows the exclusion of two possibilities: if T~ described the& +El transition, the ratio of the amplitude of T~ to the amplitudes of T~ and T~ would have to increase with increasing potassium concentration. (Potassium influences the E2 El preequilibrium.) If T corrcsponded to the dephosphorylation E2P + E2, T~ would have to be slow ( ~ 0 . s1- I ) at low potassium and fast ( = 50 S K I )at higher potassium concentrations. 6, however, shows that the shape of the signal is nearly Comparison with FIGURE independent of the potassium concentration. Thus we attribute T~ to the E I P -+ E2P conformational change. This step must be electrogenic. If, for example, only the phosphorylation were electrogenic, we would see only T , and T~ in the electrical signal. The Second Electrogenic Step: The E2K * EIH Transition
Until now we have considered only one electrogenic step. However, the lack of a steady-state current in the presence of ionophores clearly demonstrates another oppositely oriented electrogenic step. An obvious candidate is the potassium translocation betwccn E2K and EIH. Let us first consider the other possibility, that the dephosphorylation E2P -+ EZis electrogenic. Without potassium this step is rate limiting, and the concentration of E2P will bc high. For millimolar potassium concentrations the dephosphorylation rate is in the range of 50 s-'.Iy Therefore, if this step were electrogenic, it should appear in the electrical signal: there should be an additional phase with a potassiumdependent rate constant. As this is not observed, it may be excluded that the dephosphorylation is electrogenic. Therefore, we conclude that the transition from E2K to E,H is the second electrogenic step.
TRANSIENT AND STATIONARY CURRENTS GENERATED BY Na,K-ATPase-CONTAINING MEMBRANE FRAGMENTS ADSORBED TO A BLM With membrane fragments containing the pig kidney enzyme attached to a preformed BLM, it was possible to observe transient and stationary pump currents of the Na,K-ATPase after an ATP concentration jump.' The currents are sensitive to ouabain and vanadate and are dependent on the presence of Na+, Mg2+,and ATP (data not shown). Unless the conductance of a BLM is increased with ionophores, the pump current is only capacitively coupled to the outer measuring circuit. Therefore, only transient signal can be measured. If, however, the conductance of the membranes is increased by adding monensin and 1799, a stationary current in addition to the transient signal can also be measured (FIG.10). FIGUREl l a shows the dependence of peak and stationary currents on the Na+ concentration. The potassium concentration was lower than 10 kM. The presence of a stationary current under these conditions demonstrates that the Na+/Na+ exchange is electrogenic. Na+ increases the stationary current because it stimulates dephosphorylation, but with a low affinity compared to that of potassium. Potassium increased the stationary current with a &,,5 of 0.7 mM at a high ATP concentration (FIG.llb). This is due to the stimulating effect of potassium on dephosphorylation. The reduction in peak current for increasing potassium concentrations is caused by
ANNALS NEW YORK ACADEMY OF SCIENCES
180
0
100 200
300 LOO 500 600 t [msl
FIGURE 10. Electric signal in the absence and presence of ionophores. Electrolyte: 130 mM NaCl, 3 mM MgCl2,25 mM imidazole/HCl pH 7.5, T = 22°C. No potassium.
the shift of the E2K c* EINa equilibrium to the Ez form. This effect is more pronounced at low ATP levels. The concentration of free ATP can be increased significantly by a second UV flash, because the conversion ratio (released ATP/caged ATP) is in the range of 30%. Under low ( < 100 p.M) potassium conditions the stationary current is not increased by a second UV flash (FIG. 12). In the presence of 10 mM K+, however, the stationary current increases remarkably with a second concentration jump of ATP. This demonstrates the presence of an additional low affinity ATP binding site. FIGURE 13summarizes the enzymatic cycle of the Na,K-ATPase.
Assignment of the Phases of the Eleehic Signal to Partial Reactwns of the Na,K-ATPase
To describe the electric signal, at least a sum of three exponentials is necessary. In the following the three relaxation times are called I ] ,T ~ and , T ~ In . particular at a higher pH, where the photolysis of caged ATP is slow, a delay D may also be used to describe the electrical signaL2 73 has to be assigned to the characteristic time constant TO of the equivalent circ~it.2.~ T~ and T ~however, , describe the charge translocation during the enzymatic cycle. The assignment of these relaxation times to partial reactions is controversial. According to Ape11 et ab,2O the first phase (TI) is governed by electrical silent processes, including photochemical release and binding of ATP to the protein, whereas the decaying phase (Q) represented the EIP -+ E2P transition with the deocclusion of sodium. Fendler et al. ,2 however, assigned T~ to the phosphorylation or/and phosphoenzyme interconversion and TZ to the binding of ATP. Their central argument is the ATP dependence of T ~ as, shown in FIGURE 14. Recently, a detailed analysis taking into account the effect of caged ATP on ATP binding confirmed that T2 represents ATP binding under the influence of the El-caged ATP e El preequilibrium.I7J8Thus, T~represents the phosphorylation or the phosphoenzyme interconversion with sodium deocclusion.
STENGELIN el al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
181
By chymotrypsin modification of the protein, Borlinghaus et aL3excluded that the phosphorylation of El . ATP + EIP is electrogenic. Therefore, it can be concluded that the E,P + E2P transition with the deocclusion of sodium is electrogenic. This is generally accepted. A
0
Istat
Ipwk
t3A -
nA
cml
cmz 5-
o
o
o
o
o
0
o
0
0
0 A
-6
A
10A
A
-1
A A
"-8
A
A
-2
A
B
-0.-
+270pM
5pM cATP
6-500pM cATP
cATP
Ipeak
Istot nA CGl2
80
Go
----_
I
b
5
I
10
I
15 mM KCI
LO
I
20
FIGURE 11. Dependence of peak and stationary currents on Na+ and K+. (a) Na+ dependence of peak currents (circles) and stationary currents (rriungles) in the presence of monensin and the protonophore 1799. Electrolyte: Imidazole/HCI 25 mM, pH 7.5, MgC12 3 mM, K+ = 6 KM, T = 22°C. (b) Potassium dependence of peak and stationary currents. Triangles and squares: Measurement of peak currents with 5 or 500 KM caged ATP, corresponding to 1.5 and 150 pM ATP after an UV flash. Values are normalized to 100%. Circles: K+ dependence of stationary currents in the presence of monensin and 1799with 270 pM caged ATP, corresponding to 80 pM released ATP. The solid line is a fit according to a Michaelis-Menten equation, resulting in a of 0.7 mM. Electrolyte: Imidazole/HC125 mM, pH 7.5, Na+ 130 mM, MgClz 3 mM, T = 22°C.
ANNALS NEW Y O U ACADEMY OF SCIENCES
182
n* cm2
llstflash
L-
\
2-
t
1
I
1
I
FIGURE 12. Demonstration of ATP stimulation of peak currents in the presence and absence of K+.Caged ATP concentration = 50 pM corresponding to 15 pM released ATP after the first and additional 10 p,M ATP with the second flash. (a) 100 mM NaCI, 3 mM MgClz, 25 mM imidazole/HCl, pH 7.5, T = 22°C. (b) Same membrane and electrolyte as in a after the addition of 10 mM KCI.
CURRENT VOLTAGE MEASUREMENTS OF Na,K-ATPase INCORPORATED INTO PLANAR LIPID BILAYERS With the formation of artificial lipid membranes by apposition of wo lipid monolayers,ll it is possible to incorporate membrane proteins directly into a membrane separating two compartments of a cuvette.12-*4The direct incorporation is
FIGURE 13. Mechanism of the Na,K-ATPase. Loop 1 shows the Na-ATPase cycle, loop 2 the Na,K-ATPase cycle at low ATP concentrations, and loop 3 the physiological case, in which the low affinity ATP binding site is saturated.
STENGELIN el al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
183
monitored by the immediate occurrence of stationary pump currents without the help of ionophores. These currents again depend on the presence of Na+, MgZ+,and ATP and are completely blocked by 200 FM vanadate from the ATP-containing compartment and by 200 pM ouabain from the “extracellular” compartment (not shown). As incorporated and attached ATPases coexist in these experiments, the current pattern is mostly a stationary current preceded by a transient current peak (FIG.15). Under these conditions it is assumed that the ATPase molecules in the artificial membrane sense the membrane potential in the same way as they d o in biological membranes. Therefore, in this type of experiment we addressed mainly the question of the voltage dependence of the Na,K-ATPase. pH 6 2
“f??
0 [msl O
O+-----I Ims D I
iS = O.2ms
-25
0
10 20 3 40 released ATP IpMl
0
5 10 1s 20 rbleased ATPIPMI
FIGURE 14. ATP dependence of the inverse time constants T;’, 72’ and the delay D of the UV flash-induced current at pH 6.2 and 7.7. 7, and D were determined as described in Fendler et a1.l The electrolyte contained imidazoleiHCl25 mM, 130 mM NaCI, 20 mM KCI, 3 mM MgClr, T = 22°C. Different amounts of caged ATP were added. Each flash converted 10% of the caged ATP. For 7;’ the solid line is a fit to a Michaelis-Menten type concentration dependence 1 / 7 2 = ( I / T ~ ).ATP/(ATP ~,~ + K0.5) to the data. For 7;’ and D thesolidline is a horizontal line at the average value.
Until now two IV curves of the Na,K-ATPase were obtained, the first under symmetrical ionic conditions with 130 mM NaCl on both sides of the membrane (FIG. 16). The shape of the curve monotonously increases with the increasing positive potential of the ATP-containing compartment (equivalent to depolarization of the cell). Under these conditions no saturation behavior is detectable. The second curve was obtained under essentially the same conditions, with the only exception that the Na+ concentration was reduced on both sides to 20 mM, which is near the KM(Na)in the presence of 20 mM KCL4 The curve still increases monotonously but now it saturates beyond +50 mV (FIG.17).
ANNALS NEW YORK ACADEMY OF SCIENCES
184
The increase in stationary pump currents with depolarization is in accordance with electrogenic step(s) during the translocation of Na+ ions to the extracellular surface of the cell. This agrees with independent observations of the electrogenicity of sodium-transporting steps.”-2*s22 The lack of any phase of negative slope in the wide voltage range applied in both curves means that under these conditions the K+ transport does not consist of electrogenic steps. This point is still under debate, as
I
I
ATp
FIGURE 15. Pump current patterns of
a
1
the Na,K-ATPase. Electrolyte: 130 mM NaCI, 20 mM KCI, 3 mM MgC12,25 mM imidazole/HCl, pH 7.4; 1 mM DTT (all on both sides), 15 BM free ATP (only c b side), T = 22°C. (a) Schematic image of attached membrane fragments producing only capacitive currents (upper part) and incorporated pump molecules producing stationary currents (lowerpart).The normal current pattern is therefore believed to be a combined current response of attached and incorporated pump molecules. (b) Trace b: A commonly observed combination of a peak current followed by a stationary current component. Truce u: Occasionally, no incorporation occurred and only capacitive peak currents were observed. Truce c: Sometimes the number of incorporated pumps exceeded the attached ones and only the stationary current component was visible.
exist. The latter group in particular positive data22-24as well as negative claim that the binding steps of the two K+ ions to their extracellular binding sites should be electrogenic.” According to the theory,w31 a considerable voltage dependence of an electrogenic ion pump should occur in the case of a rate-limiting electrogenic step. This is in contrast to some findings that steps in the K+ branch of the Na,K-ATPase cycle are
185
STENGELIN el a!.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
3.0 2.5 2.0
1/10 1.5
0.0
-
-400 -300 -200
-100
0
100
200
300
400
FIGURE 16. IV curve without ion gradients. Electrolyte: 130 mM NaCI, 20 mM KCI, 3 mM MgC12, 25 mM imidazoleiHCl pH 7.4; 1 mM DTT (all on both sides); 15 pM free ATP/flash (cis chamber only). The guideline is a fit with two exponentials.
2.0
1.5
1/10 1.0
0.5 l
0.0
a
1
-200
-100
0 [ mvl
100
200
FIGURE 17. IV curve with low sodium concentration. Electrolyte: 20 mM NaCI, 20 mM KCI, 3 mM MgC12, 25 mM imidazole/HCl, pH 7.4; 1 mM DTT (all on both sides), 15 FM free ATP/flash (only cis side). The guideline is a fit according to a saturation curve with two exponentials. The dashed line is the guideline from FIGURE 16 for comparison.
186
ANNALS NEW YORK ACADEMY OF SCIENCES
slow and the electrogenic sodium transport is not rate limiting.32.33However, Bahinsky et aLZ4demonstrated that a fast electrogenic step could modulate the equilibrium concentration of a following rate-limiting, electroneutral step in such a way that a significant voltage dependence will occur. This model obviously also helps to explain the data presented here. The marked decrease in voltage dependence at 20 mM Na+ should therefore be due to the establishment of a slow and electroneutral step preceding the electrogenic step in the pump cycle. This new slow step following the previous rate-limiting step is much less affected by changes in the membrane potential than is the first one. Apparently, because of the sodium dependence of the effect, the sodium-binding step(s), also not rate limiting at high sodium concentrations, became slow at low sodium concentrations, implying that the sodium-binding step(s) must be electroneutral. The estimated reversal potential of both IV curves is in the range of several hundred millivolts. This agrees well with the reversal potential of -750 mV calculated from the ATP, ADP, and Pi concentrations with the assumption of one net charge transported in the absence of ion gradients (osmotic work). Our results are therefore in agreement with the commonly accepted 3 Na+/2 K+ stoichiometry of the Na,K-ATPase. REFERENCES
K., E. GRELL,M. HALJBS & E. BAMBERG. 1985. Pump currents generated by the 1. FENDLER, purified Na+K+-ATPase from kidney on black lipid membranes. EMBO J. 4 30793085. 2. FENDLER, K., E. GRELL& E. BAMBERG. 1987. Kinetics of pump currents generated by the Na,K-ATPase. FEBS Lett. 224: 83-88. R., H. J. APELL& P. LAUGER.1987. Fast charge translocation associated 3. BORLINGHAUS, with partial reactions of the NaK pump. I. Current and voltage transients after photochemical release of ATP. J. Membr. Biol. 97: 169-178. 4. NAGEL,G., K. FENDER, E. GRELL& E. BAMBERG. 1987. Na+ currents generated by the purified Na,K-ATPase on planar lipid membranes. Biochim. Biophys. Acta 901: 239249. K., E. GRELL,W. HASSELBACH & E. BAMBERG. 1987. Electrical pump currents 5. HARTUNG, generated by the ca-ATPase of sarcoplasmatic reticulum vesicles adsorbed on black lipid membranes. Biochim. et Biophys. Acta 900 209-220. 6. VAN DER HIIDEN,H. T. W. M., E. GRELL,J. J. H. H. M. DE PONT& E. BAMBERG. 1990. Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H,K-ATPase. J. Membr. Biol. 114: 245-256. 7. KAPLAN,J. H., B. FORBUSH& J. F. HOFFMANN.1978. Rapid photolytic release of adenosin-5 triphosphatase from a protected analogue: Utilization by the Na:K pump of human red blood cell ghosts. Biochemistry 17: 1925-1935. 8. SACCOMANI, G., H. B. STEWART, D. SHAW,M. LEWIN& G. SACHS.1977. Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPasecontaining vesicles by zonal centrifugation and free-flow electrophoresis technique. Biochim. Biophys. Acta 465: 311-330. 9. JORGENSEN, P. L. 1974. Isolation of NaK-ATPase. In Methods in Enzymology. S. Fleischer & L. Packer, eds.: 277-290. Academic Press. New York. M., K. FENDLER& E. BAMBERG.1992. Kinetics of charge translocation 10. STENGELIN, generated by the H,K-ATPase after an ATP concentration jump. J. Membr. Biol. Submitted. 11. MONTAL,M. & P. MUELLER.1972. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc. Natl. Acad. Sci. USA 6 9 3561-3566. E., N. A. DENCHER, A. FAHR& M. P. HEYN.1981. Transmembraneous 12. BAMBERG, incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers. Proc. Natl. Acad. Sci. USA 7 8 7502-7506.
STENGELIN e l al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
187
1990.Voltage-dependent pump currents of the sarcoplasA. & E. BAMBERG. 13. EISENRAUCH. mic reticulum Ca’+-ATPase in planar lipid membranes. FEBS Lett. 268: 152-156. 1991. Voltage dependence of the Na,KA,, E. GRELt. & E. BAMBERG. 14. EISENRAUCH, ATPase incorporated into planar lipid membranes. In J. H. Kaplan & P. De Weer, eds. The Sodium Pump: Structure, Mechanism, and Regulation. Rockefeller University Press. New York. B.. B. WALLMARK & G. SACHS.1981. The interaction of H+ and K+ with the 15. STEWART. partial reactions of gastric H,K-ATPase. J. Biol. Chem. 256 2682-2690. & G . SACHS.1983. Interaction of fluorescein isocyanate with R. J., J. MENDLEIN 16. JACKSON. the H.K-ATPase. Biochim. Biophys. Acta 731: 9-15. s. JARUSCHEWSKI, A. IIOBBS, w. ALBERS,E. BAMBEKG & E. 17. FENDLkR, K., J. FROELICH, GKELL.1991. Correlation of charge translocation with the reaction cycle of the Na,K-ATPase. I n The Sodium Pump: Recent Developments. 525-530. Society of General Physiologists Series 46 Part 2. J. H. Kaplan, ed. P. de Weer. K., S. JARUSCHEWSKI, A. HOBBS,W. ALBERS & J. P. FKOEHLICH. 1992. Presteady 18. FENDLER, state charge translocation in Na,K-ATPase from eel electric organ. Submitted to J. Gen. Physiol. 19. WALLMARK, E. RAHON, G. SACCOMANI & G. SACHS.1980.The catalytic B., H. 8. STEWART, cycle of gastric H,K-ATPase. J. Biol. Chem. 255: 513-519. H.-J., R. BORLINGHAUS & P. LAUGER.1987. Fast charge translocation associated 20. APELL, with partial reactions of the NaK pump. 11. Microscopic analysis of transient currents. J. Membr. Biol. 97: 179-191. 1986. Voltage dependence of the Na translocation by the 21. NAKAO,M. & D. C. GADSBY. Na/K pump. Nature 323: 628-630. 1986. Electrical potential accelerates A., D. E. RrCHARDs & S. J. D. KARLISH. 22. REPHA~LI. the EIP(Na)-E2P conformational transition of (Na, K)-ATPase in reconstituted vesicles. J. Biol. Chem. 261: 12437-12440. & W. D. STEIN.1987. The effect of R.. S. J. D. KARI-ISH, A. REPHAEI-I 23. GOLDSHI.~GER, membrane potential on the mammalian sodium-potassium pump reconstituted into phospholipid vesicles’. J. Physiol. (Lond.) 387: 331-355. 1988. Potassium translocation by the Na+/K+ 24. BAHINSKI, A., M. NAKAO& D. C . GADSBY. pump is voltage insensitive. Proc. Natl. Acad. Sci. USA 85: 3412-3416. 1986. Voltage dependence of the rheogenic Nat/Kt A. V. & W. SCHWARZ. 25. LAFAIRI;, ATPase in the membrane of oocytes of Xenopris larvis. J. Membr. Biol. 91: 43-51. W. & Q. Gu. 1988. Characteristics of the Na+/K+-ATPase from Towedo 26. SCHWARZ. calijiomicci expressed in Xenopus oocytes: A combination of tracer flux measurements with electrophysiological measurements. Biochim. Biophys. Acta 945: 167-174. 1991. A negative slope in R. F., L. A. VASILETS, J. LA TONA& W. SCHWARZ. 27. RAKOWSKI, the current-voltage relationship of the Na+/K+pump in Xenopus oocytes is produced by reduction of external K+.J. Membr. Biol. 121: 177-187. 28. DE WEER,P. 1984. Electrogenic pumps: Theoretical and practical considerations. I n M. P. Blaustein & M. Lieberman, eds. Electrogenic Transport: Fundamental Principles and Physiological Implications. Raven Press. New York. 29. ~t WEER,P. 1986.The electrogenic sodium pump: Thermodynamics and kinetics. In H. C. Luettgau, ed. Fortschrifte der Zoologie: Membrane control of cellular activity. Gustav Fischer Verlag. Stuttgart, New York. 30. LAUGER.P. 1984. Thermodynamic and kinetic properties of electrogenic ion pumps. Biochim. Biophys. Acta 7 7 9 307-341. 31. LAUGER,P. 1988. Electrogenic properties of the Na+/K+-pump.In The Ion PumpsStructure-Function-Regulation. W. D. Stein, ed. Alan Liss Inc. New York. 32. KARI-ISH,S. J. D. & D. W. YATES.1978. Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism. Biochim. Biophys. Acta 527: 115-130. 1. M., Y. HARA, D. E. RICHARDS & M. STEINBERG. 1987. Comparison of rates of 33. GLYNN, cation release and of conformational change in dog kidney Na,K-ATPase’. J. Physiol. (Lond.) 383: 477-485. 34. WALL MARK, B. & S. MARDH.1979. Phosphorylation and dephosphorylation kinetics of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa. J. Biol. Chem. 254: 11899-1 1902.
ANNALS NEW YORK ACADEMY OF SCIENCES
188
35.
M., F. V. VEGA& S. MARDH.1984. Effects of pH on the interaction of ligands with the H,K-ATPase purified from pig gastric mucosa. BBA 769 220-230.
LNNGSTROM,
DISCUSSION SERGIOPAPA(Institute of Medical Biochemistry, Ban, Italy): Can you elaborate on your system? Does the limited current generated by the Na+/K+ pump in the absence of K+ and by the H+/K+pump in the absence of K+ mean that the antiport is under your conditions replaced by a limited (transient) electrogenic uniport? Did you verify if ATP was hydrolyzed when the current was generated? E. BAMBERG: In both cases (Na+K+ ATPase, H+K+ATPase) in the absence of K+ the dephosphorylation is slow. Measurements of ATPase activity under the same conditions show an ATP dependence in the micromolar range, which was verified also in the BLM studies. The electrical nonstationary current reflects the transition from El to E2P. B. A. MELANDRI (Universityof Bologna, Bologna, Italy): (1) What is the quantum yield for ATP release from caged ATP and what is its dependence on concentration? (2) What is the relaxation time for the release of ATP from caged ATP? E. BAMBERG: (1) About 30% of total caged ATP is released by one laser flash, independent of concentration (for a range of 1 kM-1 mM. (2) The release of ATP goes within 1-2 ms and is strongly pH dependent. LASZLOMESZAROS (Medical College of Georgia,Augusta, GA):From the conductance values, can you give an estimate of the number of ATPase molecules in the bilayer? E. BAMBERG: Yes, from the stationary current and the turnover of the pump, a number of about 106pumps/cm2 was calculated. JACKKLEINMAN (Medical College of WBconsin, Milwauke, WI):Do your experiments rule out H+ transport by Na+ pump in the absence of Na+? E. BAMBERG: We have tried to repeat the experiments of Blostein and coworkers. We do not see a H+ current. Probably the H+ transport is so slow or small that it is buried in the noise of the system. V. SKULACHEV (Institute of Physico-Chemical Biology, Moscow, Russia): In the experiment of the ATPase incorporation into bilayer, did you add ouabain on the trans or cis side of the membrane? E. BAMBERG: Ouabain has no effect if it is added to the ATP binding site, but inhibition occurs after adding it to the tram site (opposite the ATP binding site). V. SKULACHEV: (1) ATP hydrolysis at neutral p H results in acidification of the medium. How do you exclude such an effect, especially in the presence of protonophore? What happens if pH buffer is added or if monensin is excluded? (This possibility should be considered especially carefully when K+ is absent.) E. BAMBERG:At a high buffer concentration (up to 50 mM) and after a concentration jump of ATP from caged ATP, the released H+ due to the photoreaction does not interfere with the electrical signal. Control experiments with caged ATP in the presence and absence of protein confirmed this observation. GIUSEPPE INESI(University of Maryfand, Baltimore, MD):Am I correct in assuming that the transient event is related to the prevalence of a simple intermediate species after ATP discharge, whereas the stationary event is lower due to the scrambling of intermediate states? E. BAMBERG: The transient current reflects a single turnover experiment, where the ATPases go synchronously of the enzymatic cycle through the different intermediates. The stationary compound is limited in amplitude by the slowest step in the cycle, which is in the absence of K+ the dephosphorylation.
H,K-ATPase from pig stomach and Na,K-ATPase from pig kidney were prepared as previously des~ribed.83~ ATPase-containing vesicles or membrane fragments 170
STENGELIN et al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
171
were adsorbed to a BLM (FIG. 1) as described elsewhere.'-',"' A rapid ATP concentration jump was generated by photolysis of caged ATP. The released ATP activates the ATPase and a transient current can be measured. Steady-State Current Voltage Measurements with Incorporated Na,K-ATPuse Incorporation of the Na,K-ATPase into planar lipid membranes was performed by the method of Montal and Muellerll with minor modifications, as described
rq
A
FlGURE 1. Schematic representation of the bilayer setup. (A) Teflon cell with black lipid membrane and adsorbed H,KATPase vesicles. (B) Proposed arrangement of vesicles and underlying lipid membrane. (C) Equivalent circuit diagram of the two membranes in series. G,,,represents the ohmic conductance of the combined parts of the black lipid membrane and adsorbed ATPase vesicles. G,, is the conductance of the residual vesicle membrane. C,, and C,, are the corresponding capacitances. I, designates the pump current generator.
7,K-ATPase
B
II ELM caged ATP
C
previously.I2-l4Monolayers from a solution of 2% glycerolmonooleate in hexadecane and from some microliters of a suspension of ATPase-containing membrane fragments were formed on the surface of electrolyte reservoirs in the two halves of a teflon cuvette. The electrolyte levels were raised until the monolayers fold to form a bilayer over the hole in a septum (diameter 150-200 pm), dividing the cuvette. Bilayer formation was monitored by controlling membrane conductance and capaci-
172
ANNALS NEW YORK ACADEMY OF SCIENCES
tance. The ATPase was activated by an ATP concentration jump after UV illumination of caged ATP. Current-voltage curves were obtained by applying various voltages using a constant voltage source. As every IV curve summarizes the results of several experiments with different current amplitudes, data are expressed as normalized current amplitudes I/bversus applied voltage. The currents were converted and amplified by a current-voltage converter and stored on a digital storage oscilloscope.
ELECTRIC SIGNALS GENERATED BY THE H,K-ATPase As the H,K-ATPase is an electroneutral pump, no stationary current can be observed. By separation of the H+-transporting half cycle from the K+-transporting part, however, charge translocation was observed during the El --* E2Ptransition. Adding the H,K-ATPase-containing membranes to one side of the cuvette and stirring for about 30 minutes led to adsorption of the membranes to the lipid bilayer. Release of ATP from caged ATP with an UV laser flash generated a biphasic electric signal (FIG.2a). After the addition of SCH 28080 (FIG.2b) or omeprazole (data not shown), specific inhibitors of the H,K-ATF'ase, the signal disappeared. The first phase of the signal represents the movement of a positive charge towards the BLM. The biphasic shape of the signal can be attributed to the electrical properties of the compound membrane. Asum of three exponentials ( T ; ~= 400~-~, 7i1 = lOOs-I, 73' = 10s-1) could be fitted to the transient signal. The model with three exponential components gave
a
d FIGUREZ. Inhibitionof the electric signal by SCH 28080, a specific inhibitor of the H,K-ATPase. The electrolyte contained imidazole buffer 50 mM,pH 6.14, MgClz 3 mM,EGTA 0.25 mM,DTT 0.5 mM, caged ATP 0.146 mM, and 7 = 23%: (a) without SCH 28080; and (b) after the addition of 10 FM S C H 28080.
b
+
10 pM SCH28080
20 PA
i 1
I 0-3
lo-*
Time [s]
lo-'
1oo
STENGELIN el a/.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
G = 0.26 pS/cm
173
'r
FIGURE 3. Conduction dependence of the electric signal. The electrolyte contained imidazole buffer 50 mM, pH 6.2, MgC12 3 rnM, EGTA 0.25 m M ,DTT 0.5 mM, KC1 0.5 mM, glucose 50 pM, hexokinase 1 p, caged ATP 0.26 rnM, 7 = 14%. and T = 300 K. Conductance was increased by the addition of monensin and 1799.
0.001
0.01
0.1
1
Time [ s ]
good fits and was generally used in the part of this study dealing with the H,KATPase. The conductance of the membranes was increased by adding ionophores. As in previous work,' the protonophore 1799 plus the Na+,K+/H+-exchangingcarrier monensin was used. With increasing conductance, the magnitude of the second phase of the electrical signal decreases, and at conductivities of about 3 pS/cm2 the signal becomes monophasic (FIG. 3). In contrast to that with the (Na,K)-ATPase' and the CaATPase,' no stationary current occurs either with or without K+, indicating that the pump operates electroneutrally under stationary conditions. Thus, there must be at least two oppositely directed electrogenic steps.h The basis for the following discussion is the reaction cycle shown in FIGURE4. Effect of Potassium on E2 ++ El Preequilibrium Causes a Reduction in the Electric Signal
The ion selectivity of the electric signal with respect to monovalent cations was investigated.6 Sodium ions had no effect on the signal in the range of 0-10 mM. K+ and Rb+ decreased the peak current with a of 3 mM. The effect was even more pronounced for TI+(FIG.5). With increasing potassium concentrations the magnitude of the electric signal decreased. The signal form, however, did not change (FIG.6). This can be explained if the transition from E2 to El is In this case, enzyme molecules starting in E2
174
ANNALS NEW YORK ACADEMY OF SCIENCES
ElcgATP
11
72
A
L
-e
-
73
Jm
+e
H+I
+
i
-K
1
FIGURE 4. Reaction scheme for the H,K-ATF'ase. before an ATP concentration jump produce only a minimal electric signal, because a slow step precedes the following faster reactions. Potassium shifts the E2 e El preequilibrium towards &,15J6 thus increasing the pool of enzymes that do not produce an electric signal. 72Reflects
the ATP Binding
The ATP concentration was varied in two ways: first, the caged ATP concentration was increased at a constant ATP/caged ATP conversion ratio q; second, -q was changed at a constant caged ATP concentration of 1.3 mM (FIG. 7). The peak current, Ipeok, increased with increasing ATP concentrations. Ipak was higher for identical ATP concentrations if the caged ATP concentration was lower, indicating that caged ATP inhibits the pump. T;' was independent of the ATP and
I max InA/cm21 0
Nd
0
K*
o
Rb* V
TI*
A
\ I
0
3
G
9
12
ImMl FIGURE 5. Dependence of the peak current on monovalent cations. The electrolyte contained imidazole buffer 50 mM, pH 6.0, MgC12 3 mM, caged ATP 0.1 mM, q = 30%, and T = 21°C.
STENGELIN et a/.:CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
175
caged ATP concentration. 7;' became faster for increasing ATP concentrations. For identical ATP concentrations, 7;' was slower if the caged ATP concentration is higher. T~ increased with increasing ATP concentration without a saturation up to 0.3 m M ATP. A model that takes into account the competitive binding of caged ATP to El can explain the ATP depcndcnce of 721n.37.18: k;
-
El caged ATP 7 E l k,
k- is
k:
-f
EIATP + E I P
the caged ATP dissociation rate constant. no K
+
I
c
k: and k,' are the quasi
1 m M K+
I
I
0.00 1
J
0.01
0.1
1
Time [s] FIGURE 6. Electrical signal at various K+ concentrations. The electrolyte contained imidazole buffer 50 mM, pH 6.08, MgClz 3 mM, EGTA 0.25 m M ,DTT 0.5 mM, caged ATP 0.19 mM. -q = 22%. and T = 300 K.
first-order binding-rate constants for caged ATP and ATP, respectively. They depend on the second-order rate constants k,f and k,+: k:
= k:qc,0
(1)
k,' k:(l - q)c; (2) c:!is the caged ATP concentration before the laser flash. The relevant reciprocal relaxation time is a hyperbolic function of the caged ATP
176
ANNALS NEW YORK ACADEMY OF SCIENCES
concentration, if q is held constant:
with (4) 0 0
r,-'
a
0 . 0
600 400
[s-'1
200 0
-1
72
0
t
FIGURE7. ATP dependence of the
300
'A
200
[s-l]
100
0 -1
r3
P I
A
I
A
p
A
2
a A , "
A&nv C
20
10 0
'max
A
1000
gPooo I
,
on+,
electrical signal. The electrolyte contained imidazole buffer 50 mM, pH 6.06, MgC12 3 mM, EGTA 0.25mM, D'IT 0.5 mM,T = 300 K. The ATP concentration was varied in the following way: the conversion rate of caged ATP to ATP was kept at 21% and the caged ATP concentration varied (openfigures).Then the caged ATP concentration was kept constant at 1.3 mM and the conversion rate varied by changing the laser intensity (filled figures). (a-c) The resulting time constants of a fit with three exponentials; (d) peak current.
500
For caged ATP concentrations that are large compared to h.5 ':A of (1 - q)/q:
is a linear function
As FIGURE 8 shows, this model is in accordance with the ATP/caged ATP dependence of 7 2 .
STENGELIN ef al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
FIGURE 8. Fit of the caged ATPATP dependence to the model described in the discussion. (a) 7;' shows a hyperbolic dependence with respect to the free ATP concentration. Fit parameters: b . 5 = 26.1 +M, k,,, = 256 s-I. (b) 72 is a linear function of (1 - q)/q with gradient 0.46 ms and intercept 1.86 ms.
177
b
I
Equations 1 to 6 can be used to estimate the kinetic constants k,+,k:, and k ; . TABLE1 summarizes the results of four measurements under similar conditions. Meanvalueswereky = 3 8 0 s - ' , k : = 1.7.10bs-'M-',andk,+ = 8.3. 1Obs-'M-l. rl Corresponds to the Egi TP
-+
EIP Transition
So far we have attributed 7;' to the ATP binding. In a linear reaction scheme, must be assigned to a step between the formation of ElATP and the electrogenic step or to the electrogenic step itself. TABLE2 summarizes rate constants for the EIATP E I P reaction taken from the literature at different pHs, corrected for T = 300 K. These rate constants are in a time range we can detect and should appear in our signal. A comparison of TABLE2 and the pH dependence of implies that 7;' represents the phosphorylation EIATP + EIP. FIGURE 9 shows the pH dependence of the electric signal. The pH was varied by titrating the electrolyte with HCI. The pH dependence of 7' is bell shaped with a maximum at about pH 5.8. 72 increased monotonously between pH 5 and pH 6.5. 7;'
-+
TABLE1. Kinetic Constants for ATP and Caged ATP Binding to El: Results of Different Experiments under Similar Conditions (Imidazole 50 mM, MgClz3 mM, DTT 0.5 mM, EGTA 0.25 mM. T = 300 K. DH 6.06-6.12)
HK28NO HK0341 HK0841 HK2 I N1
450 540 280 244
2.57 2.48 1.10 0.72
Mean value
379
1.72
13.6 10.0 4.9 4.7 8.29
ANNALS NEW YORK ACADEMY OF SCIENCES
178
TABLE2. Rate Constants for the El .ATP +. E I P Transition Corrected for T = 300 K 5 Wallmark34 L j u n g ~ t r o m ~ ~ 215 S K I
6
I
260 s-l
120 s-'
8
9
43 s-l
6.3 s-l
1.4 150s-1
Time constants 7;' and 7;' that are separated by a factor of about 4 at pH 5.8 show an opposite pH dependence and cannot be distinguished at about pH 6.5-7. 7;' increased with increasing pH. T~ Represents the Electrogenic ErP -+ E2P
Transition
In contrast to the interpretation of similar measurements with the NaK73 is probably not the system time constant but it describes a process of the enzymatic reaction cycle.10
FIGURE 9. pH dependence of the electrical signal. The electrolyte contained imidazole buffer 50 mM, MgC12 3 mM,EGTA 0.25 mM, DTf 0.5 mM, caged ATP 0.26 mM, = 27% T = 300 K. The solution was titrated with HCI. This figure summarizes two subsequent experiments in different pH ranges: (a) Two fast time constants for both experiments; (b) slow time constant 7;'; and (c) peak current.
STENGELIN et al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
179
If the reaction cycle for the H,K-ATPase is similar to the Albers-Post reaction scheme, T~ must be related to a process either preceding the formation of El or following the formation of ElP. The potassium dependence of the electric signal allows the exclusion of two possibilities: if T~ described the& +El transition, the ratio of the amplitude of T~ to the amplitudes of T~ and T~ would have to increase with increasing potassium concentration. (Potassium influences the E2 El preequilibrium.) If T corrcsponded to the dephosphorylation E2P + E2, T~ would have to be slow ( ~ 0 . s1- I ) at low potassium and fast ( = 50 S K I )at higher potassium concentrations. 6, however, shows that the shape of the signal is nearly Comparison with FIGURE independent of the potassium concentration. Thus we attribute T~ to the E I P -+ E2P conformational change. This step must be electrogenic. If, for example, only the phosphorylation were electrogenic, we would see only T , and T~ in the electrical signal. The Second Electrogenic Step: The E2K * EIH Transition
Until now we have considered only one electrogenic step. However, the lack of a steady-state current in the presence of ionophores clearly demonstrates another oppositely oriented electrogenic step. An obvious candidate is the potassium translocation betwccn E2K and EIH. Let us first consider the other possibility, that the dephosphorylation E2P -+ EZis electrogenic. Without potassium this step is rate limiting, and the concentration of E2P will bc high. For millimolar potassium concentrations the dephosphorylation rate is in the range of 50 s-'.Iy Therefore, if this step were electrogenic, it should appear in the electrical signal: there should be an additional phase with a potassiumdependent rate constant. As this is not observed, it may be excluded that the dephosphorylation is electrogenic. Therefore, we conclude that the transition from E2K to E,H is the second electrogenic step.
TRANSIENT AND STATIONARY CURRENTS GENERATED BY Na,K-ATPase-CONTAINING MEMBRANE FRAGMENTS ADSORBED TO A BLM With membrane fragments containing the pig kidney enzyme attached to a preformed BLM, it was possible to observe transient and stationary pump currents of the Na,K-ATPase after an ATP concentration jump.' The currents are sensitive to ouabain and vanadate and are dependent on the presence of Na+, Mg2+,and ATP (data not shown). Unless the conductance of a BLM is increased with ionophores, the pump current is only capacitively coupled to the outer measuring circuit. Therefore, only transient signal can be measured. If, however, the conductance of the membranes is increased by adding monensin and 1799, a stationary current in addition to the transient signal can also be measured (FIG.10). FIGUREl l a shows the dependence of peak and stationary currents on the Na+ concentration. The potassium concentration was lower than 10 kM. The presence of a stationary current under these conditions demonstrates that the Na+/Na+ exchange is electrogenic. Na+ increases the stationary current because it stimulates dephosphorylation, but with a low affinity compared to that of potassium. Potassium increased the stationary current with a &,,5 of 0.7 mM at a high ATP concentration (FIG.llb). This is due to the stimulating effect of potassium on dephosphorylation. The reduction in peak current for increasing potassium concentrations is caused by
ANNALS NEW YORK ACADEMY OF SCIENCES
180
0
100 200
300 LOO 500 600 t [msl
FIGURE 10. Electric signal in the absence and presence of ionophores. Electrolyte: 130 mM NaCl, 3 mM MgCl2,25 mM imidazole/HCl pH 7.5, T = 22°C. No potassium.
the shift of the E2K c* EINa equilibrium to the Ez form. This effect is more pronounced at low ATP levels. The concentration of free ATP can be increased significantly by a second UV flash, because the conversion ratio (released ATP/caged ATP) is in the range of 30%. Under low ( < 100 p.M) potassium conditions the stationary current is not increased by a second UV flash (FIG. 12). In the presence of 10 mM K+, however, the stationary current increases remarkably with a second concentration jump of ATP. This demonstrates the presence of an additional low affinity ATP binding site. FIGURE 13summarizes the enzymatic cycle of the Na,K-ATPase.
Assignment of the Phases of the Eleehic Signal to Partial Reactwns of the Na,K-ATPase
To describe the electric signal, at least a sum of three exponentials is necessary. In the following the three relaxation times are called I ] ,T ~ and , T ~ In . particular at a higher pH, where the photolysis of caged ATP is slow, a delay D may also be used to describe the electrical signaL2 73 has to be assigned to the characteristic time constant TO of the equivalent circ~it.2.~ T~ and T ~however, , describe the charge translocation during the enzymatic cycle. The assignment of these relaxation times to partial reactions is controversial. According to Ape11 et ab,2O the first phase (TI) is governed by electrical silent processes, including photochemical release and binding of ATP to the protein, whereas the decaying phase (Q) represented the EIP -+ E2P transition with the deocclusion of sodium. Fendler et al. ,2 however, assigned T~ to the phosphorylation or/and phosphoenzyme interconversion and TZ to the binding of ATP. Their central argument is the ATP dependence of T ~ as, shown in FIGURE 14. Recently, a detailed analysis taking into account the effect of caged ATP on ATP binding confirmed that T2 represents ATP binding under the influence of the El-caged ATP e El preequilibrium.I7J8Thus, T~represents the phosphorylation or the phosphoenzyme interconversion with sodium deocclusion.
STENGELIN el al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
181
By chymotrypsin modification of the protein, Borlinghaus et aL3excluded that the phosphorylation of El . ATP + EIP is electrogenic. Therefore, it can be concluded that the E,P + E2P transition with the deocclusion of sodium is electrogenic. This is generally accepted. A
0
Istat
Ipwk
t3A -
nA
cml
cmz 5-
o
o
o
o
o
0
o
0
0
0 A
-6
A
10A
A
-1
A A
"-8
A
A
-2
A
B
-0.-
+270pM
5pM cATP
6-500pM cATP
cATP
Ipeak
Istot nA CGl2
80
Go
----_
I
b
5
I
10
I
15 mM KCI
LO
I
20
FIGURE 11. Dependence of peak and stationary currents on Na+ and K+. (a) Na+ dependence of peak currents (circles) and stationary currents (rriungles) in the presence of monensin and the protonophore 1799. Electrolyte: Imidazole/HCI 25 mM, pH 7.5, MgC12 3 mM, K+ = 6 KM, T = 22°C. (b) Potassium dependence of peak and stationary currents. Triangles and squares: Measurement of peak currents with 5 or 500 KM caged ATP, corresponding to 1.5 and 150 pM ATP after an UV flash. Values are normalized to 100%. Circles: K+ dependence of stationary currents in the presence of monensin and 1799with 270 pM caged ATP, corresponding to 80 pM released ATP. The solid line is a fit according to a Michaelis-Menten equation, resulting in a of 0.7 mM. Electrolyte: Imidazole/HC125 mM, pH 7.5, Na+ 130 mM, MgClz 3 mM, T = 22°C.
ANNALS NEW Y O U ACADEMY OF SCIENCES
182
n* cm2
llstflash
L-
\
2-
t
1
I
1
I
FIGURE 12. Demonstration of ATP stimulation of peak currents in the presence and absence of K+.Caged ATP concentration = 50 pM corresponding to 15 pM released ATP after the first and additional 10 p,M ATP with the second flash. (a) 100 mM NaCI, 3 mM MgClz, 25 mM imidazole/HCl, pH 7.5, T = 22°C. (b) Same membrane and electrolyte as in a after the addition of 10 mM KCI.
CURRENT VOLTAGE MEASUREMENTS OF Na,K-ATPase INCORPORATED INTO PLANAR LIPID BILAYERS With the formation of artificial lipid membranes by apposition of wo lipid monolayers,ll it is possible to incorporate membrane proteins directly into a membrane separating two compartments of a cuvette.12-*4The direct incorporation is
FIGURE 13. Mechanism of the Na,K-ATPase. Loop 1 shows the Na-ATPase cycle, loop 2 the Na,K-ATPase cycle at low ATP concentrations, and loop 3 the physiological case, in which the low affinity ATP binding site is saturated.
STENGELIN el al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
183
monitored by the immediate occurrence of stationary pump currents without the help of ionophores. These currents again depend on the presence of Na+, MgZ+,and ATP and are completely blocked by 200 FM vanadate from the ATP-containing compartment and by 200 pM ouabain from the “extracellular” compartment (not shown). As incorporated and attached ATPases coexist in these experiments, the current pattern is mostly a stationary current preceded by a transient current peak (FIG.15). Under these conditions it is assumed that the ATPase molecules in the artificial membrane sense the membrane potential in the same way as they d o in biological membranes. Therefore, in this type of experiment we addressed mainly the question of the voltage dependence of the Na,K-ATPase. pH 6 2
“f??
0 [msl O
O+-----I Ims D I
iS = O.2ms
-25
0
10 20 3 40 released ATP IpMl
0
5 10 1s 20 rbleased ATPIPMI
FIGURE 14. ATP dependence of the inverse time constants T;’, 72’ and the delay D of the UV flash-induced current at pH 6.2 and 7.7. 7, and D were determined as described in Fendler et a1.l The electrolyte contained imidazoleiHCl25 mM, 130 mM NaCI, 20 mM KCI, 3 mM MgClr, T = 22°C. Different amounts of caged ATP were added. Each flash converted 10% of the caged ATP. For 7;’ the solid line is a fit to a Michaelis-Menten type concentration dependence 1 / 7 2 = ( I / T ~ ).ATP/(ATP ~,~ + K0.5) to the data. For 7;’ and D thesolidline is a horizontal line at the average value.
Until now two IV curves of the Na,K-ATPase were obtained, the first under symmetrical ionic conditions with 130 mM NaCl on both sides of the membrane (FIG. 16). The shape of the curve monotonously increases with the increasing positive potential of the ATP-containing compartment (equivalent to depolarization of the cell). Under these conditions no saturation behavior is detectable. The second curve was obtained under essentially the same conditions, with the only exception that the Na+ concentration was reduced on both sides to 20 mM, which is near the KM(Na)in the presence of 20 mM KCL4 The curve still increases monotonously but now it saturates beyond +50 mV (FIG.17).
ANNALS NEW YORK ACADEMY OF SCIENCES
184
The increase in stationary pump currents with depolarization is in accordance with electrogenic step(s) during the translocation of Na+ ions to the extracellular surface of the cell. This agrees with independent observations of the electrogenicity of sodium-transporting steps.”-2*s22 The lack of any phase of negative slope in the wide voltage range applied in both curves means that under these conditions the K+ transport does not consist of electrogenic steps. This point is still under debate, as
I
I
ATp
FIGURE 15. Pump current patterns of
a
1
the Na,K-ATPase. Electrolyte: 130 mM NaCI, 20 mM KCI, 3 mM MgC12,25 mM imidazole/HCl, pH 7.4; 1 mM DTT (all on both sides), 15 BM free ATP (only c b side), T = 22°C. (a) Schematic image of attached membrane fragments producing only capacitive currents (upper part) and incorporated pump molecules producing stationary currents (lowerpart).The normal current pattern is therefore believed to be a combined current response of attached and incorporated pump molecules. (b) Trace b: A commonly observed combination of a peak current followed by a stationary current component. Truce u: Occasionally, no incorporation occurred and only capacitive peak currents were observed. Truce c: Sometimes the number of incorporated pumps exceeded the attached ones and only the stationary current component was visible.
exist. The latter group in particular positive data22-24as well as negative claim that the binding steps of the two K+ ions to their extracellular binding sites should be electrogenic.” According to the theory,w31 a considerable voltage dependence of an electrogenic ion pump should occur in the case of a rate-limiting electrogenic step. This is in contrast to some findings that steps in the K+ branch of the Na,K-ATPase cycle are
185
STENGELIN el a!.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
3.0 2.5 2.0
1/10 1.5
0.0
-
-400 -300 -200
-100
0
100
200
300
400
FIGURE 16. IV curve without ion gradients. Electrolyte: 130 mM NaCI, 20 mM KCI, 3 mM MgC12, 25 mM imidazoleiHCl pH 7.4; 1 mM DTT (all on both sides); 15 pM free ATP/flash (cis chamber only). The guideline is a fit with two exponentials.
2.0
1.5
1/10 1.0
0.5 l
0.0
a
1
-200
-100
0 [ mvl
100
200
FIGURE 17. IV curve with low sodium concentration. Electrolyte: 20 mM NaCI, 20 mM KCI, 3 mM MgC12, 25 mM imidazole/HCl, pH 7.4; 1 mM DTT (all on both sides), 15 FM free ATP/flash (only cis side). The guideline is a fit according to a saturation curve with two exponentials. The dashed line is the guideline from FIGURE 16 for comparison.
186
ANNALS NEW YORK ACADEMY OF SCIENCES
slow and the electrogenic sodium transport is not rate limiting.32.33However, Bahinsky et aLZ4demonstrated that a fast electrogenic step could modulate the equilibrium concentration of a following rate-limiting, electroneutral step in such a way that a significant voltage dependence will occur. This model obviously also helps to explain the data presented here. The marked decrease in voltage dependence at 20 mM Na+ should therefore be due to the establishment of a slow and electroneutral step preceding the electrogenic step in the pump cycle. This new slow step following the previous rate-limiting step is much less affected by changes in the membrane potential than is the first one. Apparently, because of the sodium dependence of the effect, the sodium-binding step(s), also not rate limiting at high sodium concentrations, became slow at low sodium concentrations, implying that the sodium-binding step(s) must be electroneutral. The estimated reversal potential of both IV curves is in the range of several hundred millivolts. This agrees well with the reversal potential of -750 mV calculated from the ATP, ADP, and Pi concentrations with the assumption of one net charge transported in the absence of ion gradients (osmotic work). Our results are therefore in agreement with the commonly accepted 3 Na+/2 K+ stoichiometry of the Na,K-ATPase. REFERENCES
K., E. GRELL,M. HALJBS & E. BAMBERG. 1985. Pump currents generated by the 1. FENDLER, purified Na+K+-ATPase from kidney on black lipid membranes. EMBO J. 4 30793085. 2. FENDLER, K., E. GRELL& E. BAMBERG. 1987. Kinetics of pump currents generated by the Na,K-ATPase. FEBS Lett. 224: 83-88. R., H. J. APELL& P. LAUGER.1987. Fast charge translocation associated 3. BORLINGHAUS, with partial reactions of the NaK pump. I. Current and voltage transients after photochemical release of ATP. J. Membr. Biol. 97: 169-178. 4. NAGEL,G., K. FENDER, E. GRELL& E. BAMBERG. 1987. Na+ currents generated by the purified Na,K-ATPase on planar lipid membranes. Biochim. Biophys. Acta 901: 239249. K., E. GRELL,W. HASSELBACH & E. BAMBERG. 1987. Electrical pump currents 5. HARTUNG, generated by the ca-ATPase of sarcoplasmatic reticulum vesicles adsorbed on black lipid membranes. Biochim. et Biophys. Acta 900 209-220. 6. VAN DER HIIDEN,H. T. W. M., E. GRELL,J. J. H. H. M. DE PONT& E. BAMBERG. 1990. Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H,K-ATPase. J. Membr. Biol. 114: 245-256. 7. KAPLAN,J. H., B. FORBUSH& J. F. HOFFMANN.1978. Rapid photolytic release of adenosin-5 triphosphatase from a protected analogue: Utilization by the Na:K pump of human red blood cell ghosts. Biochemistry 17: 1925-1935. 8. SACCOMANI, G., H. B. STEWART, D. SHAW,M. LEWIN& G. SACHS.1977. Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPasecontaining vesicles by zonal centrifugation and free-flow electrophoresis technique. Biochim. Biophys. Acta 465: 311-330. 9. JORGENSEN, P. L. 1974. Isolation of NaK-ATPase. In Methods in Enzymology. S. Fleischer & L. Packer, eds.: 277-290. Academic Press. New York. M., K. FENDLER& E. BAMBERG.1992. Kinetics of charge translocation 10. STENGELIN, generated by the H,K-ATPase after an ATP concentration jump. J. Membr. Biol. Submitted. 11. MONTAL,M. & P. MUELLER.1972. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc. Natl. Acad. Sci. USA 6 9 3561-3566. E., N. A. DENCHER, A. FAHR& M. P. HEYN.1981. Transmembraneous 12. BAMBERG, incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers. Proc. Natl. Acad. Sci. USA 7 8 7502-7506.
STENGELIN e l al.: CHARGE TRANSLOCATION OF H,K- AND Na,K-ATPase
187
1990.Voltage-dependent pump currents of the sarcoplasA. & E. BAMBERG. 13. EISENRAUCH. mic reticulum Ca’+-ATPase in planar lipid membranes. FEBS Lett. 268: 152-156. 1991. Voltage dependence of the Na,KA,, E. GRELt. & E. BAMBERG. 14. EISENRAUCH, ATPase incorporated into planar lipid membranes. In J. H. Kaplan & P. De Weer, eds. The Sodium Pump: Structure, Mechanism, and Regulation. Rockefeller University Press. New York. B.. B. WALLMARK & G. SACHS.1981. The interaction of H+ and K+ with the 15. STEWART. partial reactions of gastric H,K-ATPase. J. Biol. Chem. 256 2682-2690. & G . SACHS.1983. Interaction of fluorescein isocyanate with R. J., J. MENDLEIN 16. JACKSON. the H.K-ATPase. Biochim. Biophys. Acta 731: 9-15. s. JARUSCHEWSKI, A. IIOBBS, w. ALBERS,E. BAMBEKG & E. 17. FENDLkR, K., J. FROELICH, GKELL.1991. Correlation of charge translocation with the reaction cycle of the Na,K-ATPase. I n The Sodium Pump: Recent Developments. 525-530. Society of General Physiologists Series 46 Part 2. J. H. Kaplan, ed. P. de Weer. K., S. JARUSCHEWSKI, A. HOBBS,W. ALBERS & J. P. FKOEHLICH. 1992. Presteady 18. FENDLER, state charge translocation in Na,K-ATPase from eel electric organ. Submitted to J. Gen. Physiol. 19. WALLMARK, E. RAHON, G. SACCOMANI & G. SACHS.1980.The catalytic B., H. 8. STEWART, cycle of gastric H,K-ATPase. J. Biol. Chem. 255: 513-519. H.-J., R. BORLINGHAUS & P. LAUGER.1987. Fast charge translocation associated 20. APELL, with partial reactions of the NaK pump. 11. Microscopic analysis of transient currents. J. Membr. Biol. 97: 179-191. 1986. Voltage dependence of the Na translocation by the 21. NAKAO,M. & D. C. GADSBY. Na/K pump. Nature 323: 628-630. 1986. Electrical potential accelerates A., D. E. RrCHARDs & S. J. D. KARLISH. 22. REPHA~LI. the EIP(Na)-E2P conformational transition of (Na, K)-ATPase in reconstituted vesicles. J. Biol. Chem. 261: 12437-12440. & W. D. STEIN.1987. The effect of R.. S. J. D. KARI-ISH, A. REPHAEI-I 23. GOLDSHI.~GER, membrane potential on the mammalian sodium-potassium pump reconstituted into phospholipid vesicles’. J. Physiol. (Lond.) 387: 331-355. 1988. Potassium translocation by the Na+/K+ 24. BAHINSKI, A., M. NAKAO& D. C . GADSBY. pump is voltage insensitive. Proc. Natl. Acad. Sci. USA 85: 3412-3416. 1986. Voltage dependence of the rheogenic Nat/Kt A. V. & W. SCHWARZ. 25. LAFAIRI;, ATPase in the membrane of oocytes of Xenopris larvis. J. Membr. Biol. 91: 43-51. W. & Q. Gu. 1988. Characteristics of the Na+/K+-ATPase from Towedo 26. SCHWARZ. calijiomicci expressed in Xenopus oocytes: A combination of tracer flux measurements with electrophysiological measurements. Biochim. Biophys. Acta 945: 167-174. 1991. A negative slope in R. F., L. A. VASILETS, J. LA TONA& W. SCHWARZ. 27. RAKOWSKI, the current-voltage relationship of the Na+/K+pump in Xenopus oocytes is produced by reduction of external K+.J. Membr. Biol. 121: 177-187. 28. DE WEER,P. 1984. Electrogenic pumps: Theoretical and practical considerations. I n M. P. Blaustein & M. Lieberman, eds. Electrogenic Transport: Fundamental Principles and Physiological Implications. Raven Press. New York. 29. ~t WEER,P. 1986.The electrogenic sodium pump: Thermodynamics and kinetics. In H. C. Luettgau, ed. Fortschrifte der Zoologie: Membrane control of cellular activity. Gustav Fischer Verlag. Stuttgart, New York. 30. LAUGER.P. 1984. Thermodynamic and kinetic properties of electrogenic ion pumps. Biochim. Biophys. Acta 7 7 9 307-341. 31. LAUGER,P. 1988. Electrogenic properties of the Na+/K+-pump.In The Ion PumpsStructure-Function-Regulation. W. D. Stein, ed. Alan Liss Inc. New York. 32. KARI-ISH,S. J. D. & D. W. YATES.1978. Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism. Biochim. Biophys. Acta 527: 115-130. 1. M., Y. HARA, D. E. RICHARDS & M. STEINBERG. 1987. Comparison of rates of 33. GLYNN, cation release and of conformational change in dog kidney Na,K-ATPase’. J. Physiol. (Lond.) 383: 477-485. 34. WALL MARK, B. & S. MARDH.1979. Phosphorylation and dephosphorylation kinetics of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa. J. Biol. Chem. 254: 11899-1 1902.
ANNALS NEW YORK ACADEMY OF SCIENCES
188
35.
M., F. V. VEGA& S. MARDH.1984. Effects of pH on the interaction of ligands with the H,K-ATPase purified from pig gastric mucosa. BBA 769 220-230.
LNNGSTROM,
DISCUSSION SERGIOPAPA(Institute of Medical Biochemistry, Ban, Italy): Can you elaborate on your system? Does the limited current generated by the Na+/K+ pump in the absence of K+ and by the H+/K+pump in the absence of K+ mean that the antiport is under your conditions replaced by a limited (transient) electrogenic uniport? Did you verify if ATP was hydrolyzed when the current was generated? E. BAMBERG: In both cases (Na+K+ ATPase, H+K+ATPase) in the absence of K+ the dephosphorylation is slow. Measurements of ATPase activity under the same conditions show an ATP dependence in the micromolar range, which was verified also in the BLM studies. The electrical nonstationary current reflects the transition from El to E2P. B. A. MELANDRI (Universityof Bologna, Bologna, Italy): (1) What is the quantum yield for ATP release from caged ATP and what is its dependence on concentration? (2) What is the relaxation time for the release of ATP from caged ATP? E. BAMBERG: (1) About 30% of total caged ATP is released by one laser flash, independent of concentration (for a range of 1 kM-1 mM. (2) The release of ATP goes within 1-2 ms and is strongly pH dependent. LASZLOMESZAROS (Medical College of Georgia,Augusta, GA):From the conductance values, can you give an estimate of the number of ATPase molecules in the bilayer? E. BAMBERG: Yes, from the stationary current and the turnover of the pump, a number of about 106pumps/cm2 was calculated. JACKKLEINMAN (Medical College of WBconsin, Milwauke, WI):Do your experiments rule out H+ transport by Na+ pump in the absence of Na+? E. BAMBERG: We have tried to repeat the experiments of Blostein and coworkers. We do not see a H+ current. Probably the H+ transport is so slow or small that it is buried in the noise of the system. V. SKULACHEV (Institute of Physico-Chemical Biology, Moscow, Russia): In the experiment of the ATPase incorporation into bilayer, did you add ouabain on the trans or cis side of the membrane? E. BAMBERG: Ouabain has no effect if it is added to the ATP binding site, but inhibition occurs after adding it to the tram site (opposite the ATP binding site). V. SKULACHEV: (1) ATP hydrolysis at neutral p H results in acidification of the medium. How do you exclude such an effect, especially in the presence of protonophore? What happens if pH buffer is added or if monensin is excluded? (This possibility should be considered especially carefully when K+ is absent.) E. BAMBERG:At a high buffer concentration (up to 50 mM) and after a concentration jump of ATP from caged ATP, the released H+ due to the photoreaction does not interfere with the electrical signal. Control experiments with caged ATP in the presence and absence of protein confirmed this observation. GIUSEPPE INESI(University of Maryfand, Baltimore, MD):Am I correct in assuming that the transient event is related to the prevalence of a simple intermediate species after ATP discharge, whereas the stationary event is lower due to the scrambling of intermediate states? E. BAMBERG: The transient current reflects a single turnover experiment, where the ATPases go synchronously of the enzymatic cycle through the different intermediates. The stationary compound is limited in amplitude by the slowest step in the cycle, which is in the absence of K+ the dephosphorylation.
