SUPPLEMENTARY MATERIAL
Availability of MgATP as Na,K-pump substrate
Since "pipette MgATP" is not necessarily equivalent to intracellular MgATP available as a substrate, six control assays were conducted with 145 mM Na+o and no added pipette ADP to assess the degree of saturation of the Na,K-ATPase with 3.7 mM MgATP ([Mg]free ~1.0 mM). Analysis of the results in terms of Qtot, Vq, and ktot at +80 mV, yielded values that were indistinguishable from those obtained with 13.2 mM MgATP (Table 2). These results indicate that even the lowest "pipette [MgATP]" used in the experiments was high enough to saturate ATP binding sites on the pump.
TABLE 2. Effect of pipette [MgATP] on charge movement parameters
[MgATP](mM) / Qtot
(fC/pF) / Vq
(mV) / k+80
(s-1)
3.7 / 26 ± 1 / -20 ± 3 / 137 ± 7
8.2 / 25 ± 1 / -23 ± 3 / 140 ± 5
13.2 / 26 ± 2 / -21 ± 5 / 131 ± 5
"Free ADP" versus MgADP
To increase [ADP]free in the pipette solution while keeping constant [Mg]total, ADP was added and [ATP]total was accordingly reduced (Table 1). However, in a series of control experiments, [ADP]total was increased and [ADP]free was reduced by manipulating [MgCl2] to test whether "free ADP" or MgADP was responsible for the observed effects. Thus, four cells were voltage-clamped with patch electrodes containing 3.0 mM MgADP (3.0 mM "free ADP") or 7.5 mM MgADP (1.5 mM "free ADP") and experiments were carried out as described in the main text. In all cases, 7.5 mM MgADP produced an effect on Vq and ktot at +80 mV that was smaller than that with 3.0 mM MgADP, as compared to zero-ADP conditions (Table 3). These results suggest that "free ADP" but not MgADP is responsible for the effects described in this paper, in agreement with reports indicating that Mg2+ would be released later in the Na,K-ATPase reaction pathway (Fukushima and Post, 1978; Campos and Beaugé, 1992, 1997).
TABLE 3. Effect of free ADP and MgADP on charge movement parameters
[ADP]free(mM) / [MgADP]
(mM) / Vq
(mV) / k+80
(s-1)
0 / 0 / -20 ± 3 / 138 ± 4
1.5 / 7.5 / -15 ± 2 / 110 ± 5
3.0 / 3.0 / -9 ± 3 / 92 ± 4
Did adenylate kinase activity affect intracellular levels of ADP?
Muscle cells contain large amounts of adenylate kinase which reversibly catalyzes the reaction 2ADP « ATP + AMP. Although pipette solutions were designed to minimize changes in [ADP] and [ATP] produced by cell metabolism, adenylate kinase activity might generate ADP from a high pipette [ATP] and residual cytosolic AMP. Conversely, high [ADP] in the pipette solution could promote synthesis of ATP by this enzyme, thus reducing the effective [ADP] present in the assays.
Adenylate kinase is competitively inhibited by a nucleotide analogue, P1,P5-di(adenosine-5’)pentaphosphate (Ap5A) with a Ki ~30 nM (Lienhard and Secemski, 1973). This inhibitor has been successfully used to study ADP effects on Na,K-ATPase from ATP-depleted red blood cells (Cavieres and Glynn, 1979; Kennedy et al., 1986). Thus, this compound was used to test whether adenylate kinase was affecting [ADP] in the present experiments. Assays were performed in ventricular myocytes superfused with 145 mM Na+o and voltage-clamped with patch electrodes containing 13.2 mM MgATP, 0.5 mM Ap5A, and no added ADP. Analysis of five complete data sets of ouabain-sensitive currents from three cells yielded the following values for the parameters shown to be a function of [ADP]: Vq = -23 ± 2 mV and k+80 = 145 ± 4 s-1. Comparison of these values with those obtained in the absence of Ap5A (-21 ± 3 mV, 141 ± 5 s-1) indicated that adenylate kinase activity was not a significant source of contaminating ADP. Three data sets from two other cells voltage-clamped in the presence of 6.5 mM MgATP, 3.0 mM ADP, and 0.5 mM Ap5A yielded parameters similar to those obtained with 3.0 mM ADP in Ap5A-free solution (not shown) ruling out an overestimation of [ADP] due to adenylate kinase activity.
Extracellular Na+ binding to a finite number of sites on the pump
Equation 5 can be modified to make Na+o binding a saturable process:
(11)
Notice that, when VM approaches -¥ (i.e. when [Na]l ® +¥), k-2 approaches a VM-independent maximum equal to k-2. KM is defined as the value of [Na]on at which k-2 equals k-2/2 at 0 mV. Equation 11 was replaced into Eq. 10 and the resulting expression was simultaneously fitted to all three curves in Fig. 5 F to yield: k2 = 144 ± 8 s-1, k-2 = 326 ± 11 s-1M-1, n = 0.90 ± 0.04, z = 0.84 ± 0.15, KM = 16 ± 3 M. The values of k2, n, and z were consistent with those found in Results. On the other hand, k-2 was twofold larger than calculated with Eq. 8. Interestingly, when this value of k-2 was used to calculate the ratio k-2/k2 and replaced as K2 in Eq. 7, values of Vq predicted from kinetics matched those obtained with steady-state charge distribution measurements. The large value of KM likely results from working with bulk [Na]o far below the K0.5 for Na+o binding.
References
Cavieres, J.D., and I.M. Glynn. 1979. Sodium-sodium exchange through the sodium pump: the roles of ATP and ADP. J. Physiol. (Lond.) 297:637-645
Fukushima, Y., and R.L. Post. 1978. Binding of divalent cations to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase. J. Biol. Chem. 253:6853-6862
Lienhard, G.E., and I.I. Secemski. 1973. P1,P5-di(adenosine-5 ')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. J. Biol. Chem. 248:1121-1123