Research ArticleION TRANSPORT

Modulation of Cl signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT

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Science Signaling  30 Oct 2018:
Vol. 11, Issue 554, eaat5018
DOI: 10.1126/scisignal.aat5018
  • Fig. 1 IRBIT interacts with and recruits kinases and phosphatases to the AID of NBCe1-B.

    (A and B) Immunoprecipitates (IP) prepared from lysates from human embryonic kidney (HEK) cells transfected with IRBIT and CaN, CaMKII, PP1, or SPAK were immunoblotted (IB) for the indicated proteins. (A) IRBIT immunoprecipitates were probed for the indicated kinases or phosphatases. Representative example of three to four similar experiments. (B) Kinase or phosphatase immunoprecipitates were probed for IRBIT. Representative example of three to four similar experiments. (C) The indicated immunoprecipitates were prepared from lysates from HEK cells expressing NBCe1-B with or without IRBIT and were immunoblotted for the indicated kinases and phosphatases. In the controls (Con), precipitating antibodies were not added. (D) The means ± SEM of IRBIT-enhanced coimmunoprecipitates were determined from six similar experiments and normalized to coimmunoprecipitation in the absence of IRBIT. (E) Lysates from HEK cells transfected with IRBIT and CaN, CaMKII, PP1, or SPAK were incubated with partially purified His-tagged NBCe1-B(1-95) or NBCe1-B(1-429) fragments, and pulldown experiments were performed. (F) The means ± SEM of the IRBIT-enhanced pulldown determined from three to four independent experiments and normalized to pulldown in the absence of IRBIT. AU, arbitrary units.

  • Fig. 2 PP1 acts on NBCe1-B(Ser65) to control Clin sensing by the 32GXXXP36 site.

    (A) Example of current time course traces in response to adding bath HCO3 and the current/voltage (I/V) at peak current. (B) HEK cells transfected with NBCe1-B and with or without IRBIT and PP1 were used to measure NBCe1-B–mediated current due to Na+-2HCO3 cotransport at 5 mM Clin. The columns show the means ± SEM. NS, not significant. (C) Current was measured in HEK cells transfected with NBCe1-B alone or with PP1, IRBIT, or IRBIT and PP1. Pipette solutions containing the indicated Cl concentrations are shown. Normalized current is shown to illustrate the effect of IRBIT on Cl sensing. (D) Current was measured in HEK cells transfected with the mutants NBCe1-B(32GP/AA36) or NBCe1-B(194GP/AA198) and with IRBIT and PP1 at pipette solutions containing 5, 40, or 140 mM Cl. The models in (C) and (D) depict the effect of the indicated condition on the state of Cl sensing. Closed GXXXP motifs indicate Cl sensing by both motifs and inhibition by Clin at high affinity, partially opened GXXXP motifs indicate loss of Cl sensing by one GXXXP motif and Cl sensing by the available motif at low affinity, and fully opened GXXXP motifs indicate complete loss of Clin sensing. The numbers in the columns and next to the symbols indicate the number of current measurements obtained from at least three independent experiments. The results are given as means ± SEM.

  • Fig. 3 SPAK acts on Ser65 in NBCe1-B to control Clin sensing by the 32GXXXP36 site.

    (A to D) Current was measured in HEK cells transfected with wild-type NBCe1-B and IRBIT (A and C), NBCe1-B(32GP/AA36) or NBCe1-B(194GP/AA198) and IRBIT (B and D), or NBCe1-B(S65E) and IRBIT (C), NBCe1-B(S65A) alone or with IRBIT (C and D), and NBCe1-B, IRBIT, and DNSPAK (A and B) and with pipette solutions containing the indicated Cl concentrations. Example of current traces at the indicated Clin concentrations and the indicated NBCe1-B mutants are shown in (A) and (D). The models in (B) to (D) depict the effect of the indicated conditions on the state of Cl sensing by the GXXXP motifs. The numbers in the columns and next to the symbols indicate the number of current measurements obtained from three to five independent transfections, and the results are given as means ± SEM.

  • Fig. 4 CaN and CaMKII regulate NBCe1-B activity, and CaN controls Clin sensing by the 194GXXXP198 motif.

    (A) Current was measured in Xenopus oocytes injected with complementary RNAs (cRNAs) encoding NBCe1-B, after NBCe1-B, H2O, CA-CaN(R392X), the indicated amount of IRBIT, and the IRBIT (LCVP/AAAA) mutant alone or together with CA-CaN. (B) Current was measured in Xenopus oocytes injected with cRNAs encoding NBCe1-B and CaMKII, IRBIT, or both. The numbers in the columns indicate the number of current measurements obtained from four independent batches of oocytes from four frogs, and the results are given as means ± SEM. (C to E) Current was measured in HEK cells transfected with NBCe1-B (C and D), NBCe1-B (32GP/AA36) or NBCe1-B(194GP/AA198) (E), and CA-CaN (C to E) or CA-CaN + PP1 (C and D). Normalized current is shown in (D). The models in (C) and (E) depict the effect of the indicated condition on the state of Cl sensing by the GXXXP motifs. The numbers in the columns and next to the symbols indicate the number of current measurements obtained from three to four independent transfections, and the results are given as means ± SEM.

  • Fig. 5 CaN acts on Ser12 in NBCe1-B to control Clin sensing by the 194GXXXP198 motif.

    (A) Effect of the S12A and S12D mutations on NBCe1-B surface expression and interaction with IRBIT (n = 3 independent experiments). (B to D) Effect of S12D and S12A mutations on the activity of NBCe1-B measured at 5 mM Clin (B) and increasing concentrations of Clin (C and D). The current in (C) is shown as normalized current in (D). (E) Example of current traces measured in cells expressing NBCe1-B(S12A) or NBCe1-B(S12D) and the indicated Clin. (F) NBCe1-B(S12A) current was measured in the presence of 5 or 140 mM pipette solution in HEK cells expressing NBCe1-B(S12A) alone or together with PP1, DN-SPAK, or CaN. Current was measured with the double mutants NBCe1-B(S12A/32GP/AA36) or NBCe1-B(S12A/194GP/AA198). The models in (C) and (F) depict the effect of the indicated condition on the state of Cl sensing by the GXXXP motifs. The numbers in the columns and next to the symbols indicate the number of current measurements obtained from three to four independent transfections, and the results are given as means ± SEM.

  • Fig. 6 IRBIT-modulated phosphorylation of Ser232, Ser233, and/or Ser235 affects NBCe1-B activity and activation by IRBIT.

    (A and B) Effect of the Ser232, Ser233, and/or Ser235 mutations on NBCe1-B surface expression and interaction with IRBIT (n = 4 independent experiments). (C and D) Current was measured with pipette solution containing 5 mM Cl and in the presence (+) or absence of IRBIT, as indicated. The cells were transfected with (C) NBCe1-B , NBCe1-B(S232A/S233A/S235A), NBCe1-B(S232D/S233D/S235D), NBCe1-A and its 3S/3A and 3S/3D mutants, NBCe1-B(Δ1-95) and its 3S/3A and 3S/3D mutants, (D) S232A or S232D, S233A or S233D, and S235A or S235D. The models in (C) and (D) depict the phosphorylation state of Ser232, Ser233, and Ser235 for the indicated mutants. Asterisk denotes P < 0.01 relative to no IRBIT. The numbers in the columns indicate the number of current measurements obtained from three to four independent transfections, and the results are given as means ± SEM.

  • Fig. 7 Ser232 affects Clin sensing by NBCe1-B.

    (A) Example of current time course traces in response to adding bath HCO3 measured in HEK cells expressing NBCe1-B, NBCe1-B + IRBIT, or the 3S/3A mutant, 3S/3A + IRBIT, and 3S/3D with pipette solution containing 5 or 140 mM Cl and the I/V at peak current. (B) Current density for NBCe1-B, the 3S/3A, and the S232A mutants in the presence (+) or absence of IRBIT was measured at 5 and 140 mM Clin. (C) Current of NBCe1-B(S233A) + IRBIT at 5, 40, and 140 mM Clin. (D) Effect of Clin on current measured with NBCe1-B + 5 μg of IRBIT, NBCe1-B(S235A) alone, or with 1 μg of IRBIT. The right plot shows the normalized current in the left plot. (E) A model of the three IRBIT-dependent NBCe1-B conformations and their properties with respect to autoinhibition, Clin sensing, and regulation of Clin sensing by the PP1/SPAK and CaN/CaMKII. The models in (B), (D), and (E) depict the phosphorylation state of Ser232, Ser233, and Ser235 and Clin sensing by the GXXXP motifs of each mutant. The numbers in the columns indicate the number of current measurements obtained from three to four independent transfections, and the results are given as means ± SEM.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/554/eaat5018/DC1

    Fig. S1. PP1 and SPAK, but not CaN, act through NBCe1-B Ser65.

    Fig. S2. CaMKII activates NBCe1-B when expressed in HEK cells.

    Fig. S3. NBCe1-B phosphorylation sites and the sites affected by IRBIT.

    Fig. S4. Structural model of NBCe1-B.

    Fig. S5. Sequence alignment of the NBC family.

    Fig. S6. Effect of S232D, S233D, and S235D mutations on NBCe1-B(Δ1-95) expression and activity.

    Fig. S7. Effect of S232X mutation on NBCe1-B expression and activity.

    Fig. S8. Effect of the S236D mutation in NBCe2-C on transport activity.

    Data file S1. MS data of HEK cells phosphoproteins in the absence of IRBIT.

    Data file S2. MS data of HEK cells phosphoproteins in the presence of IRBIT.

  • The PDF file includes:

    • Fig. S1. PP1 and SPAK, but not CaN, act through NBCe1-B Ser65.
    • Fig. S2. CaMKII activates NBCe1-B when expressed in HEK cells.
    • Fig. S3. NBCe1-B phosphorylation sites and the sites affected by IRBIT.
    • Fig. S4. Structural model of NBCe1-B.
    • Fig. S5. Sequence alignment of the NBC family.
    • Fig. S6. Effect of S232D, S233D, and S235D mutations on NBCe1-B(Δ1-95) expression and activity.
    • Fig. S7. Effect of S232X mutation on NBCe1-B expression and activity.
    • Fig. S8. Effect of the S236D mutation in NBCe2-C on transport activity.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). MS data of HEK cells phosphoproteins in the absence of IRBIT.
    • Data file S2 (Microsoft Excel format). MS data of HEK cells phosphoproteins in the presence of IRBIT.</p

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