Functional differences between alpha subunit isoforms of the rat Na,K-ATPase expressed in Xenopus oocytes

Abstract

The functional properties of the three most widely distributed alpha subunit isoforms of the Na,K-ATPase are not well known, particularly concerning the voltage dependence of their activity and cation binding kinetics. We measured the electrogenic activity generated by Na,K-ATPases resulting from co-expression of the rat alpha1, alpha2* or alpha3* subunits with the rat beta1 subunit in Xenopus oocytes; alpha2* and alpha3* are ouabain-resistant mutants of the alpha2 and alpha3 isoform, which allowed selective inhibition of the endogenous Na(+),K(+)-pump of the oocyte. In oocytes expressing the three isoforms of the alpha subunit, K(+) induced robust outward currents that were largely ouabain-sensitive. In addition, ouabain-sensitive inward currents were recorded for all three isoforms in sodium-free and potassium-free acid solutions. The very similar voltage dependence of the Na(+),K(+)-pump activity observed in the absence of extracellular Na(+) indicated a similar stoichiometry of the transported cations by the three isoforms. The affinity for extracellular K(+) was slightly lower for the alpha2* and alpha3* than for the alpha1 isoform. The alpha2* isoform was, however, more sensitive to voltage-dependent inhibition by extracellular Na(+), indicating a higher affinity of the extracellular Na(+) site in this isoform. We measured and controlled [Na(+)](i) using a co-expressed amiloride-sensitive Na(+) channel. The intracellular affinity for Na(+) was slightly higher in the alpha2* than in the alpha1 or alpha3* isoforms. These results suggest that the alpha2 isoform could have an activity that is strongly dependent upon [Na(+)](o) and [K(+)](o). These concentrations could selectively modulate its activity when large variations are present, for instance in the narrow intercellular spaces of brain or muscle tissues.

Figure 7. Apparent affinity for intracellular Na⁺

A, original current trace obtained with an sodium-deprived oocyte expressing the α3*β1 isoform of the rat Na,K-ATPase. The trace shows the successive recordings of I-V curves without and with 10 mm amiloride (pairs of arrows), and of the current induced by 10 mm K⁺ immediately thereafter, in a 10 mm Na⁺ experimental solution. Between these recordings, the oocyte was then exposed to a 100 mm Na⁺ solution in the absence of amiloride to allow for an increase in [Na⁺]_i. As indicated by the top line, the voltage was maintained at −50 mV, except for the recording of the I-V curves and during the three last sodium-loading periods, during which it was set to −100 mV to increase the rate of Na⁺ entry. Because of the large amplitude of the current flowing through the epithelial Na⁺ channel in the absence of amiloride and the relatively small size of the potassium-induced current, it was not possible to read accurately the potassium-induced current values from the paper chart. The amiloride I-V curves were recorded and analysed using the PClamp data acquisition system to define the amiloride-sensitive current reversal potential, and the potassium-induced current was recording by hand directly from the digital reading of the voltage-clamp apparatus. In this example, the potassium-induced currents were 10, 42, 112, 206, 248 and 295 nA (the last value is not visible on the trace). B, example of the potassium-activated current versus [Na⁺]_i relationship for each isoform, and the corresponding best-fitting model based on the Hill equation with a Hill coefficient of 2.5. C, mean K_1/2 parameter in the ‘box and whiskers’ representation (thick line: mean, thin line: median, bottom and top of the box: 25th and 75th percentile, vertical bars: range, circle: outlier). The maximal current obtained from the fit was 255 ± 36 nA (n = 12), 238 ± 68 nA (n = 7), and 186 ± 17 nA (n = 16) for the α1, α2* and α3* groups, respectively. The difference in the mean K_1/2 parameter between the α1 and α2* was statistically significant (t (degrees of freedom = 17): 2.1098, P≤ 0.05). The other differences were not significant.

Figure 1. Potassium-induced current due to the endogenous Na,K-ATPase and after its inhibition by pre-exposure to 200 nm ouabain in the incubation solution

Original current recordings in oocytes injected with the βsubunit of the rat Na,K-ATPase alone after overnight exposure to a potassium-free solution without ouabain (A) or from a different oocyte incubated in a solution containing 200 nm ouabain (B). I-V curves recorded before and after the addition of 10 mm potassium gluconate (10K) to a previously potassium-free solution. No ouabain was present at the time of measurement. C, mean I-V curves of the potassium-induced current (I-V in the presence of 10 mm K⁺; the mean of the I-V curve in the potassium-free solution before and after) in 10 oocytes without ouabain pre-exposure (Co, open symbols) showing the electrogenic activity of the endogenous Na⁺/K⁺-pump, and in 17 (8 non-injected and 9 injected with the β subunit alone) oocytes pre-exposed to 200 nm ouabain (black symbols). Measurements were performed the first time 1–2 min after the oocytes had been removed from the 0.2 mm ouabain-containing incubation solution and placed in the control experimental solution, which contained no ouabain: (1 min) and repeated 5–6 min (6 min) and 10–12 min (11 min) later to demonstrate the very slow dissociation rate constant (k_off) of ouabain from the Xenopus oocyte endogenous Na,K-ATPase.

Figure 2. Potassium-activated and ouabain-sensitive currents

A and B, original current recordings obtained during exposure to increasing concentrations of K⁺ (0, 0.02, 0.1 0.5 and 5.0 mm) and to 2 mm ouabain with 5.0 mm K⁺ in a sodium-free experimental solution (0K). The traces in A and B were obtained with oocytes expressing the α1 β1 and the α2*β1 isoforms of the rat Na,K-ATPase, respectively. The holding potential was −50 mV and I-V curves were recorded under each condition at the time indicated by the arrows. C, I-V relationship for the current induced by four different concentrations of K⁺ recorded in the example given in B (α2*β1 isoform). D, relationship between the potassium-induced current to the K⁺ concentration for five membrane potentials in the same set of data (trace in B).

Figure 3. Voltage dependence of the ouabain-sensitive currents (A and C) and potassium-activated currents (B and D) measured in sodium-rich solutions (A and B) and in sodium-free solutions (C and D) in oocytes expressing the α1, α2* and α3* isoform together with the β1 subunit of the rat Na,K-ATPase

In all cases the current values are normalized to the amplitude of the current induced by the highest K⁺ concentration at a membrane potential of 0 mV. For the experiments shown in the sodium-rich solution (A and B), the mean currents induced by 10 mm K⁺ were 201 ± 41 nA (α1, n = 11), 282 ± 41 nA (α2*, n = 10) and 234 ± 32 nA (α3*, n = 10). For the experiments in the sodium-free solution (C and D), the mean currents induced by 5 mm K⁺ were 299 ± 4 nA (α1, n = 17), 261 ± 41 nA (α2*, n = 15) and 174 ± 20 nA (α3*, n = 15).

Figure 4. Ouabain-sensitive inward current in a sodium-free, low-pH solution

A, the current trace recorded at −50 mV in sodium-free solutions with an oocyte expressing the α1 β1 isoform of the rat Na,K-ATPase. The activation of the Na⁺/K⁺-pump current by 5 mm K⁺ was first recorded in a solution at pH 7.4. Change to a pH 6.0, potassium-free solution induced an inward current that could be largely inhibited by 2 mm ouabain. B, the mean values of the ouabain-sensitive current (i.e. the difference between the I-V curves recorded without and with ouabain (arrows 1 and 2 in A, respectively). All of the values are expressed relative to the current induced by 5 mm K⁺ at pH 7.4. The mean normalization values were 201 ± 41 nA (n = 11), 282 ± 41 nA (n = 10) and 234 ± 32 nA (n = 10) for the α1, α2* and α3* groups, respectively.

Figure 5. Voltage dependence of the activation of the current by external K⁺

A, sodium-free solution. B, 100 mm Na⁺ solution. K_1/2 values were obtained by fitting the Hill equation to the set of five current amplitudes measured at five values of [K⁺]_o for each oocyte and at each membrane potential. The mean K_1/2 values as a function of membrane potential are shown in A (sodium-free solution) and B (100 mm Na⁺ solution). One example of the relationship between [K⁺]_o and the induced current at five different membrane potentials is given in D of Fig. 2.

Figure 6. Effect of [Na⁺]_o on the potassium-activated current

A, original current recordings showing the current induced by a [K⁺]_o of 1 mm in the presence of extracellular solutions containing 0, 30 and 100 mm[Na⁺]_o (effected by N-methyl-d-glucamine replacement). This recording was obtained with an oocyte expressing the α3*β1 isoform of the rat Na,K-ATPase. B, mean potassium-induced current expressed as relative to the current induced by 1 mm K⁺ measured at 0 mV in the sodium-free solution. The mean normalization value was 160 ± 25 nA (n = 12), 165 ± 25 nA (n = 12) and 154 ± 24 nA (n = 12) for the α1, α2*, and α3* groups, respectively. While the α1 and α3* isoforms present a rather similar voltage-dependent inhibition by external Na⁺, this inhibition was clearly more pronounced for the α2* isoform.