Fig. 1. FXYD7 is expressed specifically in brain. A 10 µg aliquot of total RNA from different rat tissues (A) or rat brain regions (B) was migrated on agarose gels and transferred onto nitrocellulose prior to hybridization with an FXYD7 cDNA probe. A 50 µg aliquot of protein from microsomes of rat brain regions or rat kidney (C) was subjected to SDS–Tricine gel electrophoresis and western blot analysis with an FXYD7 antibody.

Fig. 2. FXYD7 is present in both neurons and glial cells. Double immunostaining of rat brain sections with an affinity-purified N-terminal FXYD7 antibody and with either synaptophysin, a neuronal marker, or GFAP, an astroglial marker. Confocal analysis shows FXYD7 immunoreactivity in green (A, D, G and J), and synaptophysin (B and E) and GFAP (H and K) in red. Co-localization appears in yellow (C, F, I and L). Shown are low (200×) (A–C and G–I) and high (1000×) (D–F and J–L) magnifications. Similar results were obtained with a C-terminal FXYD7 antibody.

Fig. 3. FXYD7 is a post-translationally modified, type I protein. (A) Mouse FXYD7 cRNA was translated and labeled with ³⁵[S]methionine in a reticulocyte lysate in the absence (lane 1) or presence (lane 2) of canine pancreatic membranes, or injected into Xenopus oocytes prior to metabolic labeling with ³⁵[S]methionine for 24 h (lane 3) followed by a 48 h chase period (lane 4). After denaturing immunoprecipitations with an FXYD7 antibody of in vitro translated samples or digitonin extracts of oocytes, samples were subjected to SDS–Tricine gel electrophoresis and revealed by fluorography. In addition, proteins from in vitro translation assays (lane 5), from microsomes of FXYD7 cRNA-injected oocytes (50 µg) (lane 6) or from rat brain microsomes (50 µg) (lane 7) were migrated on SDS–Tricine gels and subjected to western blot analysis with an FXYD7 antiserum (1:5000). (B) Oocytes were injected or not (ni) with FXYD7 or N-flag FXYD7 cRNA (2 ng). Upper panel: 3 days after injection, microsomes were prepared from metabolically labeled oocytes, immunoprecipitated with an FXYD7 antibody and the immunoprecipitates migrated on SDS–Tricine gels. Lower panel: 3 days after cRNA injection, intact oocytes were subjected to a radioimmunolabeling assay using a ¹²⁵I-labeled FLAG antibody. (C) Oocytes were injected with FXYD7 cRNA, metabolically labeled for 6 h and subjected to a 48 h chase. Microsomes were immunoprecipitated with an FXYD7 antibody, and the immunoprecipitates bound to Sepharose beads were treated with or without O-glycosidase and/or neuraminidase as described in Materials and methods, before gel migration. (D) Oocytes were injected with wild-type or threonine mutant FXYD7 cRNA, metabolically labeled for 6 h and subjected to 24 and 48 h chase periods in the absence (lanes 1–24) or presence of BFA (lanes 25–28). Microsomes were immunoprecipitated under denaturing conditions with an FXYD7 antibody. a = no modified threonine (14 kDa), b = one modified threonine (15 kDa), c = two modified threonines (18 kDa) and d = three modified threonines (19 kDa). In the sequence of FXYD7, the bar indicates the position of the FXYD motif, the boxed region indicates the transmembrane segment, and the arrowheads indicate Thr3, Thr5 and Thr9, which were subjected to mutations.

Fig. 4. FXYD7 associates specifically with Na,K-ATPase α–β1 isozymes after co-expression in Xenopus oocytes and with α1–β1 isozymes in the native brain tissue. Oocytes were injected with FXYD7 cRNA (0.4 ng) together with rat Na,K-ATPase α isoforms (α1, α2 or α3; 7–10 ng) cRNAs and rat β1 (1 ng) or human β2 (1 ng) cRNAs. After injection, oocytes were metabolically labeled for 24 h and subjected to 24, 72 and 120 h chase periods. Microsomes were immunoprecipitated under non-denaturing conditions with an FXYD7 antibody (A) and under denaturing conditions with an Na,K-ATPase α antibody (B). Indicated are the positions of Na,K-ATPase α-subunits, fully glycosylated (fg) β-subunits and FXYD7. * = artifactual bands only observed in FXYD7 immunoprecipitations under non-denaturing conditions. (C) Microsomes from rat brain or kidney were either loaded directly on an SDS–Tricine gel (lanes 1 and 3) or first immunoprecipited under non-denaturing conditions with an Na,K-ATPase α antibody (lanes 2 and 4). After western blotting, FXYD7 was revealed with an FXYD7 antibody. (D) Western blot analysis of kidney and brain microsomes, which were either loaded directly on a 5–13% polyacrylamide gel (50 µg; lanes 1, 3, 6, 8, 11 and 13) or subjected first to non-denaturing immunoprecipitations with an FXYD7 antibody (lanes 2, 4, 7, 9, 12 and 14) or with a pre-immune serum (control, lanes 5, 10 and 15). After protein transfer, the blot was probed with isoform-specific α1 (lanes 1–5), α2 (lanes 6–10) or α3 (lanes 11–15) antibodies. The position of α-subunits is indicated. * = non-specific band probably due to heavy chains of antibodies.

Fig. 5. FXYD7 modulates the transport properties of Na,K-ATPase. Oocytes were injected with FXYD7 cRNA (2 ng) together with rat Na,K-ATPase α isoforms (α1, α2* or α3*, 7–10 ng; * = oubain resistant) and rat β1 (1 ng) cRNAs. The voltage dependence of the external K⁺ activation constant (K_1/2 K⁺) of Na,K-ATPase isozymes was measured in the presence (A–C) or absence (D–F) of 90 mM external Na⁺. FXYD7 had a significant (P < 0.05) effect on the K⁺ activation of α1–β1 and α2–β1 but not of α3–β1 isozymes over the whole potential range in the presence and absence of external Na⁺. The effect of external Na⁺ (G–I) on ouabain-sensitive currents (Ip) was determined by comparing Ip in the presence of external Na⁺ (Ip +Na⁺ ext) with those in its absence (Ip –Na⁺ ext). Ip was set arbitarily to 1 at 10 mV. Open squares, Na,K-ATPase alone; closed squares, Na,K-ATPase + FXYD7. Data are means ± SE of 20–40 oocytes from 4–6 different batches.

Fig. 6. Threonine modifications of FXYD7 are not involved in the association with Na,K-ATPase or in the K⁺ effect of FXYD7 on Na,K-ATPase. (A) Oocytes were injected with wild-type FXYD7 or the triple threonine mutant (T3A/T5A/T9A) cRNAs (0.4 ng) together with rat Na,K-ATPase α1 (10 ng) and rat β1 (1 ng) cRNAs. After injection, oocytes were metabolically labeled for 24 h and subjected to a 48 h chase period. Microsomes were immunoprecipitated under non-denaturing conditions with an FXYD7 antibody. Indicated are the positions of Na,K-ATPase α-subunits, core (cg) and fully (fg) glycosylated β-subunits, and of wild-type and mutant FXYD7. (B) Oocytes were injected with wild-type FXYD7 or the triple threonine mutant (T3A/T5A/T9A) cRNAs (2 ng) together with rat Na,K-ATPase α1 (10 ng) and rat β1 (1 ng) cRNAs. Three days after injection, K⁺ activation constants (K_1/2 K⁺) were determined in the presence of 90 mM external Na⁺. Open squares, Na,K-ATPase alone; closed squares, Na,K-ATPase + FXYD7; closed circles, Na,K-ATPase + T3A/T5A/T9A mutant. Data are means ± SE of 10–13 oocytes from two different batches.