The role of the third extracellular loop of the Na+,K+-ATPase alpha subunit in a luminal gating mechanism

Abstract

Na+,K+-ATPase is responsible for maintaining the cross-membrane Na+ and K+ gradients of animal cells. This P-type ATPase works via a complex transport cycle, during which it binds and occludes three intracellular Na+ ions and then two extracellular K+ ions, which it then releases on the other side of the membrane. The cation pathway through the protein, and the structures responsible for occluding cations inside the protein, have not yet been definitely identified. We used cysteine mutagenesis to explore the accessibility and the role of five conserved residues in the short third extracellular loop, between the fifth and the sixth transmembrane helices. The P801C and L802C mutants were not affected by extracellular sulfhydryl reagents. The presence of cysteine residues at three positions (G803C, T804C and V805C) conferred sensitivity to omeprazole, a known inhibitor of the gastric proton pump, and to [2-(trimethylammonium)-ethyl]methanethiosulphonate bromide (MTSET). The effects of omeprazole and MTSET were modulated by the presence of extracellular K+, indicating that the accessibility of these positions depended on the conformational state of the protein. MTSET binding to cysteine at position 803 partially inhibited the Na+,K+-pump function by decreasing its affinity towards extracellular K+, suggesting a restriction of the access of extracellular K+ ions to their binding sites. In contrast, MTSET binding to cysteine at position 805 partially inhibited the Na+,K+-pump function by reducing its maximum turnover rate, probably by slowing a rate-limiting conformational change. These residues occupy positions that are critical for either the cation pathway or the conformational modifications.

Figure 1. Effect of cysteine mutations on the K⁺-activated and ouabain-sensitive currents

A and B, original current recordings from oocytes expressing the C111S (A) and the G803C (B) mutants. The membrane voltage was clamped at −50 mV, except for six series of short voltage steps. The oocytes were exposed to a K⁺-free, pH 6.0 solution, and to a solution containing 10 mm K⁺ in the presence and in the absence of 2 mm ouabain, making it possible to determine the K⁺-activated current and the ouabain-sensitive current in the presence of 10 mm K⁺ and in a K⁺-free solution at pH 6.0. C, original current recordings during a series of 125 ms voltage steps from −130 to +30 mV, recorded first in the presence of 10 mm K⁺ (1), then with 10 mm K⁺ and 2 mm ouabain (2); the difference between these currents (2–1) gave the value of the ouabain-sensitive current. This example was taken from an oocyte expressing the C111S mutant. D, Na⁺,K⁺-pump current–voltage (I–V) curve and the effect of an acid extracellular pH. The I–V relationship of the K⁺-induced current (IK, ○), of the ouabain-sensitive current in the presence of K⁺ (Iouab, •) at pH 7.4, and the ouabain-sensitive current at pH 6.0 in the absence of K⁺ (Iouab pH 6, ▴) were recorded in experiments as illustrated in A and B. The K⁺-induced current at pH 5.0 (IK pH 5, ▵) was recorded in another series of experiments described in Fig. 2. All current values are normalized to the K⁺-induced current at −50 mV and pH 7.4 for each oocyte. Error bars represent s.e.m. Error bars are smaller than symbol size in some cases.

Figure 2. Effect of acid pH and omeprazole on cysteine mutants of the Na⁺,K⁺-pump

A and B, original current recordings in an oocyte expressing the G803C mutant of the α1 subunit of Bufo marinus Na⁺,K⁺-ATPase. The holding membrane potential was held at −50 mV, apart from a series of short voltage steps. The effect of omeprazole was tested by measuring the K⁺-induced outward current before and after a 4 min exposure to 60 μm omeprazole. A, one example in which omeprazole was applied in the absence of extracellular K⁺; B, another example in which omeprazole exposure was performed in the presence of 10 mm extracellular K⁺. C, conformation-dependent effect of omeprazole on Na⁺,K⁺-pump current. Arithmetic means ± s.e.m. of K⁺-induced currents recorded at −50 mV and pH 7.4 in oocytes expressing the test cysteine mutants, and the C111S ‘control’ mutant. All current values were expressed in terms of the K⁺-induced current measured before exposure to omeprazole for each oocyte. The number of measurements was between 4 and 11 for each condition. Error bars represent s.e.m. *P < 0.001 versus C111S (ANOVA and Bonferroni post test); #P < 0.001 for the comparison between 10 mm K⁺ and K⁺-free solution (two-way ANOVA and Bonferroni post test).

Figure 3. Effects of MTSET on Na⁺,K⁺-ATPase cysteine mutants

A, conformation-dependent effects of [2-(trimethylammonium)-ethyl]methanethiosulphonate bromide (MTSET) on Na⁺,K⁺-pump current. Means ± s.e.m. of K⁺-induced currents at −50 mV in oocytes expressing the test cysteine mutants and the ‘control’ C111S mutant. The currents measured after MTSET exposure were expressed in terms of the currents measured before exposure to MTSET for each oocyte. MTSET was perfused for 2 min at a concentration of 150 μm, either with 10 mm external K⁺ to shift the equilibrium towards the E1 conformation (black bars), or without K⁺ (grey bars) to favour the E2P conformation of the Na⁺,K⁺-ATPase. Between 4 and 11 measurements were carried out for each condition. Error bars represent s.e.m. *P < 0.001 versus C111S (ANOVA and Bonferroni post test); #P < 0.001 for the comparison between 10 mm K⁺ and K⁺-free solution (two-way ANOVA and Bonferroni post test). B and C, original current recording showing the effect of a prolonged exposure to a high concentration (1 mm) of MTSET in an oocyte expressing the G803C (B) or the V805C mutant (C). The holding potential was maintained at −50 mV. The current trace shows the activation of the Na⁺,K⁺-pump current by 10 mm K⁺, and the effect of adding 1 mm MTSET for a 3 min exposure. MTSET induced rapid (t_¹/2 < 30 s), but incomplete inhibition of the K⁺-induced current, with a residual current equal to about 40–60% of its initial value. This effect of MTSET was not reversible.

Figure 4. Effect of MTSET on the voltage-dependent kinetics of activation by extracellular K⁺

A, original current recording showing the effect of MTSET in an oocyte expressing the V805C mutant of the α1 subunit of Bufo marinus Na⁺,K⁺-ATPase. The K⁺ concentration was increased in steps from 0 to 0.3, 1.0, 3.0 and 10 mm. Adding 1 mm MTSET to the 10 mm K⁺ solution reduced the outward current. The half-life of the decrease was less than 30 s. After a 2 min exposure, the K⁺-induced current was measured again by exposure to the same external K⁺ concentrations. I–V curves were recorded for each K⁺ concentration. The values of the K⁺-induced current at each concentration were used to calculate the maximum K⁺-induced current (I_max) and the K⁺-activation constant (K_¹/2K⁺), as described in Methods. B, the three upper plots represent the I_max, and the three lower plots represent the K⁺-activation constant (K_¹/2K⁺) as a function of the membrane potential for the control C111S (n = 8) and for two cysteine mutants, G803C (n = 12) and V805C (n = 16). The K_¹/2K⁺ values were obtained, as in the example shown in A, by recording I–V curves in increasing K⁺ concentrations before and after exposure to MTSET. The curves with the open symbols represent the measurements before MTSET perfusion, and those with the filled symbols represent the measurements after MTSET exposure. Error bars represent s.e.m. Error bars are smaller than symbol size in some cases.

Figure 5. Structural model of the third extracellular loop

Using a model constructed by homology with the sarcoplasmic and endoplasmic calcium ATPase (SERCA) structure (PDB 1EUL) (Horisberger et al. 2004), this figure shows the structure of the third extracellular loop between the transmembrane (TM)5 and TM6 helices and the neighbouring TM2 and TM4 helices. The TM7, TM8, TM9 and TM10 helices are not shown, but they would be in front of those illustrated. The extracellular side of the membrane is at the top and the view is roughly parallel to the membrane. The TM helices 2, 4, 5 and 6 are shown as cylinders. The red point indicates the approximate position of cation-binding site II, close to the unwound portion of TM4. The five residues mutated in the present study are shown in CPK representation. P801 and L802 (ice-blue colour) have their side chains pointing towards the inside of the protein. G803, shown in purple, has no side chain, but the position of the side chain of a cysteine substituted at this position would point outwards, as indicated by the double purple dotted line. T804 and V805 form the first turn of the TM6 helix. T804 points towards TM4, while the side chain of V805 points towards TM2.