Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

Abstract

A family of small, single-span membrane proteins (the FXYD family) has recently been defined based on their sequence and structural homology. Some members of this family have already been identified as tissue-specific regulators of Na,K-ATPase (NKA). In the present study, we demonstrate that phospholemman (PLM) (FXYD1), so far considered to be a heart- and muscle-specific channel or channel-regulating protein, associates specifically and stably with six different alpha-beta isozymes of NKA after coexpression in Xenopus oocytes, and with alpha1-beta, and less efficiently with alpha2-beta isozymes, in native cardiac and skeletal muscles. Stoichiometric association of PLM with NKA occurs posttranslationally either in the Golgi or the plasma membrane. Interaction of PLM with NKA induces a small decrease in the external K+ affinity of alpha1-beta1 and alpha2-beta1 isozymes and a nearly 2-fold decrease in the internal Na+ affinity. In conclusion, this study demonstrates that PLM is a tissue-specific regulator of NKA that may play an essential role in muscle contractility.

Fig 1.

Association of PLM with NKA isozymes in Xenopus oocytes. (A) Oocytes were injected with 8 ng of rat NKA α1, α2, or α3 cRNA and 1 ng β1 or β2 cRNA, or with 8 ng SERCA2a cRNA together with 1 ng PLM cRNA, metabolically labeled for 24 h, and subjected to a 48-h chase period. After the pulse–chase period, microsomes were prepared and immunoprecipitated with an α- (lanes 1–12) or SERCA-antibody (lanes 13 and 14) under nondenaturing conditions before subjecting to SDS/PAGE. Indicated are the positions of PLM (≈13 kDa), NKA α isoforms, SERCA2a and fully (fg) and core glycosylated (cg) β isoforms. (B) Microsomes were directly loaded on a SDS/Tricine gel. (C) Oocytes were injected with NKA α1, β1, and PLM cRNA and metabolically labeled for 24 h before microsomes were prepared and immunoprecipitations were performed with an α- (lane 1) or a PLM-antibody (lane 2) under nondenaturing conditions. (D) After injection with NKA α1, β1, and PLM cRNA, oocytes were metabolically labeled in the absence (lane 1) or presence (lanes 4 and 7) of BFA and subjected to 24- and 48-h chase periods in the absence (lanes 2, 3, 5, and 6) or presence (lanes 8 and 9) of BFA. Microsomes were prepared and immunoprecipitated with an α-antibody under nondenaturing conditions. (E) Quantification of data shown in D. Represented is the ratio PLM/α as a function of the chase period; open circles, data from D, lanes 1–3; closed circles, data from D, lanes 4–6; closed triangles, data from D, lanes 7–9.

Fig 2.

Association of PLM with NKA isozymes in native tissues. (A) Aliquots of rat brain, rat kidney, or bovine sarcolemma microsomes were migrated on SDS/Tricine gels and subjected to Western blotting. Blots were probed with an α-antibody (anti-KETYY, Upper) or an α2-specific antibody (anti-HERED, Lower). (B) Aliquots of bovine sarcolemma microsomes were suspended in a solution containing 25 mM imidazole (pH 7.5), 1 mM EDTA, and 10 mM RbCl plus 5 mM ouabain and solubilized at 0°C with 1 mg/ml of C₁₂E₁₀. Samples were either loaded directly on SDS/Tricine gels (lanes 1 and 3) or first immunoprecipitated with an α1- (lane 2) or α2-specific (lane 4) antibody under nondenaturing conditions. After Western blotting, PLM was revealed with PLM-antibodies. (C) Aliquots of rat kidney or skeletal muscle microsomes were migrated on SDS/Tricine gels and subjected to Western blotting. Blots were probed with a PLM-antibody. (D) Aliquots of microsomes of kidney (lanes 1, 2, 5, and 6) or rat skeletal muscle (lanes 3, 4, 7, and 8) were either directly loaded on a gel (lanes 1, 3, 5, and 7) or first immunoprecipitated with a PLM-antibody under nondenaturing conditions (lanes 2, 4, 6, and 8). After Western blotting, α1 (lanes 1–4) and α2 isoforms (lanes 5–8) were revealed with specific antibodies. For unknown reasons, α1 isoforms from muscle (lane 3) migrated slightly faster than α1 from kidney (lane 1) or α1 coimmunoprecipitated with PLM-antibodies (lane 4). (E) Aliquots of sarcolemma microsomes were suspended in the presence of either 20 mM Tris⋅HCl, 10 mM RbCl plus 5 mM ouabain, or 20 mM NaCl plus 0.1 mg/ml of oligomycin before solubilization as in B. Samples were immunoprecipitated (lanes 2, 4, and 6) or not (lanes 1, 3, and 5) with a α1-specific antibody and subjected to Western blotting. Blots were probed with α- (Upper) or PLM-antibodies (Lower). (F) Aliquots of sarcolemma microsomes were suspended in the presence of 10 mM RbCl plus 5 mM ouabain and solubilized with the indicated concentrations of C₁₂E₁₀. Immunoprecipitation and Western blotting were as in E.

Fig 3.

PLM modulates the apparent K⁺ affinity of NKA isozymes. Oocytes were injected with PLM cRNA (1 ng) together with rat NKA α isoforms (α1, α2*, 10 ng) and rat β1 (1 ng) cRNAs. The voltage dependence of the external K⁺-activation constant (K_1/2K⁺) of NKA α1–β1 (A and C) or α2–β1 (B and D) isozymes was measured in the presence (A and B) or absence (D and E) of 90 mM external Na⁺. PLM had a significant (P < 0.05) effect on the K⁺ activation of α1–β1 and α2–β1 isozymes over the whole potential range in the presence of external Na⁺. Open triangles, NKA alone; closed squares, NKA plus PLM. Data are means ± SE of 11–17 oocytes from two to three different batches.

Fig 4.

PLM decreases the apparent Na⁺ affinity of NKA isozymes. Oocytes were injected with rat NKA α1 and β1 (lanes 1 and 2) or α2* and β1 (lanes 3 and 4) cRNAs without (lanes 1 and 3) or with PLM (lanes 2 and 6) cRNA in the presence of cRNAs of the rat epithelial Na⁺ channel subunits (α, β, γ, 0.3 ng each), and incubated for 3 days. Apparent affinities for intracellular Na⁺ shown in A were determined as described in Materials and Methods from Na⁺-activation curves shown in B. Open circles, NKA α1–β1; closed circles, NKA α1–β1 plus PLM. PLM had a significant effect (P < 0.05) on the apparent Na⁺ affinity of each NKA isozyme tested. (Inset) Maximal pump currents (I_max). Data are means ± SE of 5–11 oocytes from two to three different batches.