Artigas P et al. (2023), A Na pump with reduced stoichiometry is up-regu...

XB-ART-60458

Proc Natl Acad Sci U S A 2023 Dec 26;12052:e2313999120. doi: 10.1073/pnas.2313999120.

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A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.

Artigas P , Meyer DJ , Young VC , Spontarelli K , Eastman J , Strandquist E , Rui H , Roux B , Birk MA , Nakanishi H , Abe K , Gatto C .

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Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na+,K+-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two β variants, in addition to the salinity-controlled α2KK. These β subunits permitted heterologous expression of the α2KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK's Lys308 helps to maintain high affinity for external K+ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K+-congener 86Rb+, we prove that double-lysine-substituted pumps transport 2Na+ and 1 K+ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.

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Species referenced: Xenopus laevis
GO keywords: sodium channel activity

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	Fig. 1. (A) Post-Albers canonical Na+, K+-ATPase catalytic cycle. The pump transports one charge per cycle as it transports 3 Na+ in exchange for 2 K+ while harnessing the energy from ATP hydrolysis by alternating between phosphorylated and dephosphorylated forms of two major conformations E1 and E2. The red dotted box encloses the states transited by the pump in the absence of external K+ in the presence of Na+, when it produces the voltage-dependent transient charge movement. (B) Alignment of the TM4 and TM5 regions of various NKA α subunits showing the two lysines present in Artemia’s α2KK (third line), where canonical NKAs have asparagines. (C and D) Extracellular view of the ion-binding site region showing ion-coordinating sidechains and N333 (Xenopus α1 numbering) in the structures of the pig NKA formed by α1β1γ, with 3 Na+ ions bound in E1 (13) (C) and of the shark NKA formed by α1β1FXYD10 with bound K+ in E2 (14) (D). (E) Box plot of fold-change of Artemia’s NKA isoforms in adult shrimp at high salinities. Data points from 5 (α3NN, β1 & β2) or 6 (α1NN & α2KK) biological replicates are shown as circles. The box edges are the quartiles, the line dividing the box is the median, the mean is an open square. A Kruskal–Wallis test indicates that only α1NN at 2 M (P = 0.006) and 4 M (P = 0.01) and α2KK (P = 0.0001) at 4 M, are ≠ than 1 (no change).
	Fig. 2. Structure of Artemia’s α2KKβ2. (A) Overall cryo-EM structure of Artemia’s α2KK in the AlF4 inhibited E2 state without bound ions viewed from the membrane plane showing the α (multicolored) and β subunits (yellow). Intracellular A-, P-, and N-domains are colored pink, green, and cyan. The membrane position is indicated by the yellow shade. (B and C) Density map in the region surrounding the ion-binding sites (B) and in the P domain, where AlF4− binds to the P-type ATPase conserved aspartic acid (C). (D) Zoomed-in view of the ion binding site region of the α2KK structure viewed from the extracellular side, approximately perpendicular to the membrane plane, a similar orientation as the ion-bound canonical structures shown in Fig. 1C.
	Fig. 3. Electrophysiological characterization of Artemia’s α2KKβ2 NKAs. (A) Representative traces in NMG+ solution at −50 mV, from oocytes injected with cRNA to form Xenopus α1β3 (Top) or Artemia’s α2KKβ2 (Bottom) NKAs. Addition of K+ activated outward currents in a concentration-dependent manner. Vertical deflections in the traces correspond to application of 100 ms-long voltage pulses used to obtain the K+-induced currents at different voltages to measure the half-maximal activating concentration. (B) K0.5,K obtained from Hill fits to the K+ concentration dependence of the steady-state current, as a function of the applied voltage obtained in the presence (solid symbols) or absence of Na+ (as in A, open symbols) for oocytes expressing Xenopus α1β3 (black circles) Artemia’s α2KKβ2 (green squares) or the double lysine mutant mimicking α2KK, Xenopus α1KKβ3 (blue triangles). The Hill fits had Hill coefficient (shared between all voltages) were nH = 1.20 ± 0.09 for Xsα1β3, nH = 1 (fixed) for α2KKβ2 and nH =1.06 ± 0.08 for Xsα1KKβ3 in NMG, and nH = 1.55 ± 0.09 (n = 13) for Xsα1β3, nH = 1 (fixed) for α2KKβ2 and nH = 0.86 ± 0.09 (n = 5) for Xsα1KKβ3. (C) Ouabain-sensitive transient currents (current without ouabain – current after ouabain) measured in the presence of 125 mM Na+ when voltage pulses were applied from −50 mV to the indicated voltages, in two oocytes expressing Xenopus α1β3 (Top) or Artemia’s α2KKβ2 (Bottom). (D) Q-V curve plotting the integral of the current signal when the voltage pulse is turned off as a function of the applied voltage for the same constructs in B. Line plots correspond to Boltzmann equations fitted to the data from individual experiments (Methods) with slope factors shared between all Boltzmann fits to different experiments (±SEM from global fit) kT/zeq = 35 ± 0.3 mV (n = 18) for Xsα1β3, kT/zeq = 64 ± 2.3 mV (n = 9) for α2KKβ2; kT/zeq = 49 ± 1.3 mV (n = 7) for Xsα1KKβ3, and kT/zeq = 44 ± 2.6 mV (n = 5) for α2NNβ2 and the midpoint voltage given in Table 1. The IC50 for inhibition of α2KK-mediated K+-induced currents by the specific inhibitor ouabain was 135 ± 90 μM (SD, n = 14) thus allowing separation of exogenous from endogenous currents by preincubation in 1 μM ouabain (Methods).
	Fig. 4. Intracellular Na+ affinity and stoichiometry of α1KKβ3. (A) Representative traces from two patches held at 0 mV. Application of 4 mM MgATP at different intracellular Na+ concentrations induced larger outward NKA currents as Na+ concentration is raised. Sharp vertical deflections correspond to application of 25 ms pulses ranging from −140 to +40 mV. (B) Average NKA current as a function of Na+ from three patches for each mutant. Line plots represent the Hill equations with parameters K0.5,Na = 3.3 ± 0.25, nH = 1.6 ± 0.1 for wild-type Xsα1β3 (black circles) and K0.5,Na =10.2 ± 2.1 nH =1.3 ± 0.1 (black circles) for Xsα1KKβ3 (blue triangles). The three individual experiments were fitted with a shared Hill coefficient. Error bars are SEM from the fit. (C) Representative traces from oocytes expressing wild-type Xsα1β3 (Top) or Xsα1KKβ3 (Bottom), held at −50 mV bathed by NMG. Application of 1 mM 86Rb+ over the oocytes reversibly induced outward current. The current integral gives the charge extruded by the oocytes. After current deactivation, the total 86Rb+ uptake by each oocyte was measured in a scintillation counter, indicated for individual experiments. (D) Bar graph summarizing the average ratio of Rb+ uptake/charge extruded from the indicated number of oocytes (from two oocyte batches for each mutant). The total uptake was 1.00 ± 0.06 nanomoles in oocytes expressing WT pumps, 465 ± 39 picomoles, 283 ± 54 picomoles in oocytes expressing N333K/N785K, and 17 ± 4 picomoles (n = 12) in impaled uninjected oocytes pretreated with ouabain. Non-NKA-mediated uptake (i.e., uptake in uninjected oocytes) was subtracted from the uptake measured in injected oocytes in the same batch of oocytes to obtain the NKA-mediated uptake (Methods).

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Abe, Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations. 2021, Pubmed