a Detection of the Ion Transport of the Na,K-ATPase Reconstituted in Lipid Vesicles

As mentioned above, Oxonol VI can be applied to detect the ion transport of the Na,K-ATPase reconstituted in lipid vesicles. In proteoliposomes containing a high concentration of K⁺ ions and a low concentration of Na⁺ ion, the extravesicular addition of ATP in the presence of Mg²⁺ and Na⁺ ions activates the enzyme molecules reconstituted with the ATP-binding site facing outward (Fig. 33). As a consequence, an inside-positive membrane po-tential is generated due to the electrogenic transport. In response, Oxonol VI molecules ac-cumulate in the intravesicular aqueous space according to a Nernst equilibrium, leading to an increased adsorption of the dye in the inner lipid monolayer and to a concomitant in-crease in fluorescence intensity (Fig. 34).

Figure 33. Upon addition of ATP in the extravesicular medium, the enzyme molecules with the ATP-binding site facing outward are activated. As a consequence, an inside-positive transmembrane potential is generated due to the electrogenic transport. In response, Oxonol VI molecules accumulate in the vesicle membrane, leading to a fluorescence increase.

The proteoliposomes prepared as described in 2.2 contain 70 mM K2SO4 and 5 mM Na2SO4. Sulfate is chosen as primary anion because it produces a significantly lower leak current than chloride (152). The experiments have been performed in a Perkin-Elmer LS 50B fluorescence spectrophotometer set at the same conditions described in 2.4.1a.

Materials

 Tris (MP Biomedicals, UltraPure)

 Imidazole (Merck, buffer substance, ACS)

 EDTA (Merck, Titriplex® II, for analysis, ACS)

 MgSO₄ heptahydrated (Merck, for analysis)

 Na₂SO₄ anhydrous (Merck, for analysis, ACS)

 ATP magnesium salt (Sigma)

 Oxonol VI (Molecular Probes)

 EtOH (Merck, for spectroscopy)

 H₂SO₄ (Backer, 95-97%)

 Sodium orthovanadate (Sigma, >90%) Solutions

 Buffer: 25 mM imidazole, 1 mM EDTA, pH 7.2 (H₂SO₄)

 1 M MgSO₄

 25 μM Oxonol VI in EtOH

 2.5 M Na₂SO₄

 1.5 M Tris/H2SO4, pH 7.0

 0.5 M ATP

 0.2 M Sodium orthovanadate, pH 7.5 (HCl)

Procedure

The experiments are performed at 20 ± 0.5 °C.

1- A cuvette made of special optical glass (Hellma, type 109.004F-OS) is filled with 1 ml of Buffer containing 2.5 mM MgSO4, 25 mM Na2SO4, and 100 mM Tris/H2SO4. The addition of Tris/H₂SO₄ provides an ion strength equivalent to the one of the intravesicular solution. The cuvette is equilibrated for 10 min inside the instrument to stabilize the desired temperature.

2- At the beginning of the experiment, 25 nM Oxonol VI is added to the cuvette.

3- After 2 min, a volume of vesicles corresponding to 80 μg/ml of lipid is added. Con-sidering an average diameter of 110 nm (113), this corresponds to about 5^.10¹¹ cles. A fluorescence increase is observed due to the insertion of the dye in the vesi-cles membrane.

4- When a stable steady-state is reached, 2.5 mM ATP is added to the cuvette. The ad-dition activates the enzyme molecules reconstituted with the ATP-binding site facing outwards. The enzymes pump three Na⁺ ions into and two K⁺ ions out of the vesi-cles, building up an inside-positive transmembrane potential that causes an increase of the fluorescence signal. The signal reaches another steady-state at elevated mem-brane potentials, when the pump current is compensated by the leak current due to the membrane conductance.

5- To evaluate the specific conductance of the membrane, 5 mM orthovanadate is add-ed at the end of the experiment. As a result, the fluorescence signal decreases be-cause of the transmembrane voltage decays due to the membrane conductance. The time constant, τ, of the decrease is equal to Cm/Gm, where Cm is the specific capaci-tance and Gm the specific conductance of the membrane. With Cm ~ 1 µF/cm², Gm = 1/τ (152).

At the end of each experiment, the cuvette is washed with 0.5 % Hellmanex II to remove any trace of lipids. The detergent is then rinsed carefully with distilled water.

To allow the comparison between different experiments, the fluorescence changes are normalized with respect to the fluorescence level after the addition of the vesicles, when the dye inserts inside the vesicles membrane. The normalized fluorescence increase can be fitted with a single exponential function (Eq. 5).

Equation 5 F_normF_min (F_max F_min)(1e^t) with

Fnorm = normalized fluorescence

F_min = normalized fluorescence level before ATP addition

Fmax = normalized fluorescence level of the steady-state after ATP addition τ = time constant of the normalized fluorescence increase after ATP addition

Figure 34. Detection of the enzyme-generated inside-positive transmembrane potential with Oxonol VI.

Oxonol VI can be used to investigate the Na⁺-binding affinity in the E₁ conformation of the Na,K-ATPase reconstituted in lipid vesicles. In this condition, it is not possible to use the dye RH421 in equilibrium-titration experiments as described in 2.4.1a because of the ex-tremely low density of enzyme molecules in the vesicle membrane. To evaluate the Na⁺ -binding affinity of the enzyme in the E₁ conformation, the enzyme-mediated generation of the electric potential across the vesicle membrane is detected as function of the extravesicular (= cytoplasmic) sodium concentration. During the initial phase of the experi-ments, before a considerable membrane potential is build up and the intravesicular potassi-um concentration becomes limiting, the enzyme activity is controlled by the rate-limiting step of the transport cycle, which is Na⁺-binding in the E1 conformation at low extravesicular sodium concentrations. Since the initial slope of the fluorescence signal is proportional to the initial enzyme activity, the Na⁺-dependence of the initial slope of the signal provides directly the Na⁺-binding affinity in the E1 conformation. In the various ex-periments,the ionic strength is kept constant by the addition of Tris/H2SO4.

The initial slope of the signal, corresponding to the derivative of the signal with respect to time at t = 0, is given by Fmax/τ, as can be derived by the following considerations. The vesicle membrane can be represented by an equivalent circuit diagram with the capacitance, C_m, and the leak conductance, λL of the lipid bilayer and the enzyme represented as a current generator, I_P.

According to basic physics these elements may be linked up in the following way:

dt C dU dt

I  dQ  _m

where Q is the electric charge on the capacitor, U the electric potential across the membrane, and I the current flowing across the membrane. The net current through the vesicle rate. Combining the equations above leads to the inhomogeneous differential equation,

C U

solved with the boundary condition, valid at long times, t → ∞,

 



I U

I_{P L} _L

where U∞ is the transmembrane voltage in the stationary phase, when the pump current is compensated by the leak current. The solution of the differential equation is

) voltage in the steady state, U∞, and the time constant, τ, of the voltage increase.

 well as the derivative of the fluorescence signal with respect to time at t = 0 is proportional to the derivative of the transmembrane voltage with respect to time at t = 0.

0 0

As a consequence, the initial slope of the normalized fluorescence signal is proportional to the ratio of the maximum amplitude, F_max, and the time constant, τ, of the normalized

The proportionality constant, K^#, depends on the properties of each specific vesicle prepara-tion and is invariable within the same preparaprepara-tion. Therefore, the results obtained with the same preparation can be directly compared, at least within a time period in which no aging processes affect the enzyme activity. Fitting the initial slope of the normalized signal versus the extravesicular sodium concentration with the Hill function (Eq. 6) allows the evaluation of the Na⁺-binding affinity in the E1 conformation of the enzyme reconstituted in lipid vesi-cles.

dF = initial slope of the normalized fluorescence signal

0 max

dF = initial slope at saturating Na⁺ concentrations [Na] = concentration of Na⁺ ions

K1/2 = half-saturating Na⁺ concentration n = Hill coefficient

The normalization of the experiments, as well as the volume and drift corrections, has been performed with the program Drifter. The fitting procedure has been performed with the data elaboration program FigP 2.98. The error is expressed as SEM.

CHAPTER 3

RESULTS

3.1 Extension of the Methods Based on the Styryl Dye RH421 to