The WNK/SPAK and IRBIT/PP1 pathways in epithelial fluid and electrolyte transport

Abstract

Fluid and electrolyte homeostasis is a fundamental physiological function required for survival and is associated with a plethora of diseases when aberrant. Systemic fluid and electrolyte composition is regulated by the kidney, and all secretory epithelia generate biological fluids with defined electrolyte composition by vectorial transport of ions and the obligatory water. A major regulatory pathway that immerged in the last several years is regulation of ion transporters by the WNK/SPAK kinases and IRBIT/PP1 pathways. The IRBIT/PP1 pathway functions to reverse the effects of the WNK/SPAK kinases pathway, as was demonstrated for NBCe1-B and CFTR. Since many transporters involved in fluid and electrolyte homeostasis are affected by PP1 and/or calcineurin, it is possible that WNK/SPAK and IRBIT/PP1 form a common regulatory pathway to tune the activity of fluid and electrolyte transport in response to physiological demands.

FIGURE 1. The known domains of the WNKs, SPAK/OSR1, and IRBIT

Known domains are marked only when first mentioned. For the WNKs, proline-rich domains (PRD) are in green, the coiled-coil domains (CCD) are in orange, the kinase domains are in red, the autoinhibitory domains (AID) are in purple, and the SPAK/OSR1 binding motifs [R/K]Fx[V/I] are in magenta. For the SPAK/OSR1, the proline- and arginine-rich domain (P/ARD) is in green, the kinase domain is in red, the serine-rich (S) motif is in purple, the WEW-containing domain that binds Cab39 is in pink, and the conserved COOH-terminal domain (CCT) is in brown. For IRBIT, the protein phosphatase 1 (PP1) binding motif is in green, the PEST domain is in red, the CCD is in orange, and the PDZ motif is in turquoise.

FIGURE 2. IRBIT antagonizes inhibition of NBCe1-B and CFTR activity and surface expression by the WNKs and SPAK

A and C: NBCe1-B activity as the Na⁺-dependent recovery from an acid load (A) and the whole cell CFTR current (C). B and D: the total and surface expression of NBCe1-B (B) and CFTR (D). The transporters were expressed in HeLa cells alone or with the indicated mutants of WNKs and SPAK and with or without IRBIT. WNK^11–119 is the NH₂ terminus 119 residues of WNK1 that do not include the kinase domain; the notation KD notes kinase-dead for both the WNKs and SPAK. The examples shown illustrate the reversal by IRBIT of the reduced surface expression and activity of the transporters by the WNKs and SPAK. The results were taken from Ref. .

FIGURE 3. WNKs and SPAK inhibit and IRBIT stimulates pancreatic duct fluid secretion

Sealed pancreatic ducts in primary culture were treated with the indicated WNKs or SPAK siRNAs or the PP1 inhibitor tautomycin (A) or si-IRBIT (B) and were used to measure stimulated fluid secretion. Knockdown of the inhibitors WNKs and SPAK enhanced secretion, whereas inhibition of PP1 that is required to reverse the effect of the WNK/SPAK and knockdown of the activator IRBIT reduced secretion. The results were taken from Refs. , .

FIGURE 4. Model of the NH₂ terminus 400 residues of NBCe1-B

Model prediction and construction is given in the text. Residues 1–62 that include the IRBIT binding domain are in orange, residues 62–100 are in blue, and the domain predicted with high homology confidence is in green.

FIGURE 5. Model for regulation of fluid and HCO₃⁻ secretion by the WNK/SPAK and IRBIT/PP1 pathways

In the resting state, SPAK is scaffold by WNK, which recruits SPAK to the HCO₃⁻ transporters. SPAK phosphorylates NBCe1-B, Slc26a6, and CFTR (indicated with red arrows and red -P) to sequester most of them in intracellular organelles (blue arrows), NBCe1-B in organelles next to the basolateral membrane, and Slc26a6 and CFTR likely in the same organelles next to the luminal membrane. In the absence of cytoplasmic IP₃ IRBIT is bound to the IP₃ receptors that in secretory cells are clustered at the apical pole. This results in low secretory activity. When the cells are stimulated with an IP₃-generating agonist, IP₃ binds to the IP₃ receptors to dissociate IRBIT that now recruits PP1 to the transporters to dephosphorylate them. The organelles with the dephosphorylated transporters that are complexed with IRBIT now fuse with the plasma membrane to insert NBCe1-B in the basolateral membrane and Slc26a6 and CFTR in the luminal membrane. IRBIT remains bound to a specific site in each of the transporters in a mode similar to binding of IRBIT to the NH₂ terminus autoinhibitory domain of NBCe1-B. Insertion of the transporters in the plasma membrane and their activation by IRBIT results in stimulation of fluid and HCO₃⁻ secretion.