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Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis.

Terker AS, Zhang C, Erspamer KJ, Gamba G, Yang CL, Ellison DH - Kidney Int. (2016)

Bottom Line: Also, chloride inhibited WNK4 within the range of distal cell chloride concentration.Mutation of a previously identified WNK chloride-binding motif converted WNK4 effects on SPAK from inhibitory to stimulatory in mammalian cells.Disruption of this motif in WNKs 1, 3, and 4 had different effects on NCC, consistent with the three WNKs having different chloride sensitivities.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology & Hypertension, Department of Medicine, Oregon Health & Science University, Portland, Oregon, USA.

No MeSH data available.


Related in: MedlinePlus

Effects of mutating the WNK Cl--sensing motif on SPAK and NCC in HEK cellsa) Western blot for pSPAK/pOxSR1 on HEK cells transfected with empty vector, WT WNK4, WNK4 L322F L324F (WNK4 LLFF), or kinase-dead WNK4 LLFF K186M. WT WNK4 reduced pSPAK/pOxSR1 abundance and WNK4 LLFF increased it. WNK4 LLFF K186M reduced pSPAK/pOxSR1 abundance to levels similar to WT WNK4. All effects were significant (p<0.05) by one-way ANOVA with Dunnett's multiple comparisons test. b-d) Western blots for pNCC-T53 in HEK cells expressing NCC and b) WT WNK1 or WNK1 L369F L371F, c) WT WNK3 or WNK3 L295F L297F, or d) WT WNK4 or WNK4 L322F L324F. LLFF mutations in WNK1 and WNK4 increased pNCC abundance. p<0.05 by unpaired t-test for both. WNK3 LLFF did not affect pNCC differently than WT WNK3. e) Bar chart comparing relative effects of the different WNK LLFF mutants versus their WT forms. p<0.05 by two-way ANOVA. Representative images are shown. f) Cartoon suggesting how K+ intake affects NCC. When plasma [K+] is low, chloride effluxes from cells, activating WNK4 kinase, which phosphorylates SPAK and therefore NCC. When plasma [K+] is high, higher intracellular chloride concentration inhibits WNK4 kinase activity so it cannot activate SPAK or NCC. Note that, for clarity, the cartoon shows phosphorylation events as ‘all or none’. These effects are likely highly graded in vivo.
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Figure 4: Effects of mutating the WNK Cl--sensing motif on SPAK and NCC in HEK cellsa) Western blot for pSPAK/pOxSR1 on HEK cells transfected with empty vector, WT WNK4, WNK4 L322F L324F (WNK4 LLFF), or kinase-dead WNK4 LLFF K186M. WT WNK4 reduced pSPAK/pOxSR1 abundance and WNK4 LLFF increased it. WNK4 LLFF K186M reduced pSPAK/pOxSR1 abundance to levels similar to WT WNK4. All effects were significant (p<0.05) by one-way ANOVA with Dunnett's multiple comparisons test. b-d) Western blots for pNCC-T53 in HEK cells expressing NCC and b) WT WNK1 or WNK1 L369F L371F, c) WT WNK3 or WNK3 L295F L297F, or d) WT WNK4 or WNK4 L322F L324F. LLFF mutations in WNK1 and WNK4 increased pNCC abundance. p<0.05 by unpaired t-test for both. WNK3 LLFF did not affect pNCC differently than WT WNK3. e) Bar chart comparing relative effects of the different WNK LLFF mutants versus their WT forms. p<0.05 by two-way ANOVA. Representative images are shown. f) Cartoon suggesting how K+ intake affects NCC. When plasma [K+] is low, chloride effluxes from cells, activating WNK4 kinase, which phosphorylates SPAK and therefore NCC. When plasma [K+] is high, higher intracellular chloride concentration inhibits WNK4 kinase activity so it cannot activate SPAK or NCC. Note that, for clarity, the cartoon shows phosphorylation events as ‘all or none’. These effects are likely highly graded in vivo.

Mentions: In cultured cells and Xenopus oocytes, WNK4 has been observed commonly to inhibit SPAK (or oxidative stress response kinase, OxSR1; sometimes called OSR1) and NCC activity. This is in contrast to WNK1 and WNK3, which typically stimulate SPAK and NCC activity in the same model conditions. WNK4 inhibition of NCC activity in oocytes has been shown to require its ability to interact with other WNKs via its HQ domain.18 Thus, Bazua-Valenti and colleagues suggested that, in oocytes, WNK4 is inactivated by ambient [Cl-] and thereby exerts a dominant-negative effect.22 To test this hypothesis, we mutated the WNK4 Cl--sensing domain that is homologous to the WNK1 domain.8 Although wild-type WNK4 decreased pSPAK in HEK cells, consistent with prior reports, WNK4 L322F L324F increased it (Fig 4a, Supplemental Fig 2). The effects of WNK4 L322F L324F were kinase-dependent because mutation of the putative catalytic lysine (WNK4 L322F L324F K186M) returned pSPAK abundance to levels similar to that observed with WT WNK4 (Fig 4a). Mutating the Cl--sensing motif in the three different WNKs was also shown to have different effects on NCC phosphorylation. Consistent with the Cl-sensitivity reported by Piala et al.8, expression of the WNK1 mutant (WNK1 L369F L371F) increased pNCC by 140%, compared to expression of WT WNK1 (Fig 4b, e). In contrast, expression of the WNK3 Cl--sensing mutant (WNK3 L295F L297F) had little effect on pNCC abundance compared to expression of WT WNK3 (Fig 4c, e), supporting our observation (Fig 3) that WNK3 is the least sensitive to [Cl-]. Expression of WNK4 L322F L324F, on the other hand, increased pNCC abundance by 339% of that resulting from WT WNK4 expression (Fig 4d, e). Thus, the effects of WNK4 chloride-sensing mutations were substantially larger than those of WNK1 and WNK3 (Fig 4e).


Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis.

Terker AS, Zhang C, Erspamer KJ, Gamba G, Yang CL, Ellison DH - Kidney Int. (2016)

Effects of mutating the WNK Cl--sensing motif on SPAK and NCC in HEK cellsa) Western blot for pSPAK/pOxSR1 on HEK cells transfected with empty vector, WT WNK4, WNK4 L322F L324F (WNK4 LLFF), or kinase-dead WNK4 LLFF K186M. WT WNK4 reduced pSPAK/pOxSR1 abundance and WNK4 LLFF increased it. WNK4 LLFF K186M reduced pSPAK/pOxSR1 abundance to levels similar to WT WNK4. All effects were significant (p<0.05) by one-way ANOVA with Dunnett's multiple comparisons test. b-d) Western blots for pNCC-T53 in HEK cells expressing NCC and b) WT WNK1 or WNK1 L369F L371F, c) WT WNK3 or WNK3 L295F L297F, or d) WT WNK4 or WNK4 L322F L324F. LLFF mutations in WNK1 and WNK4 increased pNCC abundance. p<0.05 by unpaired t-test for both. WNK3 LLFF did not affect pNCC differently than WT WNK3. e) Bar chart comparing relative effects of the different WNK LLFF mutants versus their WT forms. p<0.05 by two-way ANOVA. Representative images are shown. f) Cartoon suggesting how K+ intake affects NCC. When plasma [K+] is low, chloride effluxes from cells, activating WNK4 kinase, which phosphorylates SPAK and therefore NCC. When plasma [K+] is high, higher intracellular chloride concentration inhibits WNK4 kinase activity so it cannot activate SPAK or NCC. Note that, for clarity, the cartoon shows phosphorylation events as ‘all or none’. These effects are likely highly graded in vivo.
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Figure 4: Effects of mutating the WNK Cl--sensing motif on SPAK and NCC in HEK cellsa) Western blot for pSPAK/pOxSR1 on HEK cells transfected with empty vector, WT WNK4, WNK4 L322F L324F (WNK4 LLFF), or kinase-dead WNK4 LLFF K186M. WT WNK4 reduced pSPAK/pOxSR1 abundance and WNK4 LLFF increased it. WNK4 LLFF K186M reduced pSPAK/pOxSR1 abundance to levels similar to WT WNK4. All effects were significant (p<0.05) by one-way ANOVA with Dunnett's multiple comparisons test. b-d) Western blots for pNCC-T53 in HEK cells expressing NCC and b) WT WNK1 or WNK1 L369F L371F, c) WT WNK3 or WNK3 L295F L297F, or d) WT WNK4 or WNK4 L322F L324F. LLFF mutations in WNK1 and WNK4 increased pNCC abundance. p<0.05 by unpaired t-test for both. WNK3 LLFF did not affect pNCC differently than WT WNK3. e) Bar chart comparing relative effects of the different WNK LLFF mutants versus their WT forms. p<0.05 by two-way ANOVA. Representative images are shown. f) Cartoon suggesting how K+ intake affects NCC. When plasma [K+] is low, chloride effluxes from cells, activating WNK4 kinase, which phosphorylates SPAK and therefore NCC. When plasma [K+] is high, higher intracellular chloride concentration inhibits WNK4 kinase activity so it cannot activate SPAK or NCC. Note that, for clarity, the cartoon shows phosphorylation events as ‘all or none’. These effects are likely highly graded in vivo.
Mentions: In cultured cells and Xenopus oocytes, WNK4 has been observed commonly to inhibit SPAK (or oxidative stress response kinase, OxSR1; sometimes called OSR1) and NCC activity. This is in contrast to WNK1 and WNK3, which typically stimulate SPAK and NCC activity in the same model conditions. WNK4 inhibition of NCC activity in oocytes has been shown to require its ability to interact with other WNKs via its HQ domain.18 Thus, Bazua-Valenti and colleagues suggested that, in oocytes, WNK4 is inactivated by ambient [Cl-] and thereby exerts a dominant-negative effect.22 To test this hypothesis, we mutated the WNK4 Cl--sensing domain that is homologous to the WNK1 domain.8 Although wild-type WNK4 decreased pSPAK in HEK cells, consistent with prior reports, WNK4 L322F L324F increased it (Fig 4a, Supplemental Fig 2). The effects of WNK4 L322F L324F were kinase-dependent because mutation of the putative catalytic lysine (WNK4 L322F L324F K186M) returned pSPAK abundance to levels similar to that observed with WT WNK4 (Fig 4a). Mutating the Cl--sensing motif in the three different WNKs was also shown to have different effects on NCC phosphorylation. Consistent with the Cl-sensitivity reported by Piala et al.8, expression of the WNK1 mutant (WNK1 L369F L371F) increased pNCC by 140%, compared to expression of WT WNK1 (Fig 4b, e). In contrast, expression of the WNK3 Cl--sensing mutant (WNK3 L295F L297F) had little effect on pNCC abundance compared to expression of WT WNK3 (Fig 4c, e), supporting our observation (Fig 3) that WNK3 is the least sensitive to [Cl-]. Expression of WNK4 L322F L324F, on the other hand, increased pNCC abundance by 339% of that resulting from WT WNK4 expression (Fig 4d, e). Thus, the effects of WNK4 chloride-sensing mutations were substantially larger than those of WNK1 and WNK3 (Fig 4e).

Bottom Line: Also, chloride inhibited WNK4 within the range of distal cell chloride concentration.Mutation of a previously identified WNK chloride-binding motif converted WNK4 effects on SPAK from inhibitory to stimulatory in mammalian cells.Disruption of this motif in WNKs 1, 3, and 4 had different effects on NCC, consistent with the three WNKs having different chloride sensitivities.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology & Hypertension, Department of Medicine, Oregon Health & Science University, Portland, Oregon, USA.

No MeSH data available.


Related in: MedlinePlus