Limits...
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 [Cl-] on in vitro WNK kinase activity using SPAK as a substratea) Effects of increasing [Cl-] on kinase activity on the WNK1 kinase domain (WNK1KD) and the WNK4 kinase domain (WNK4KD) in vitro. SPAK was used as the substrate. WNK4KD activity was inhibited at much lower [Cl-] than WNK1KD. For WNK4KD, N=4 for all data points except log[Cl-]=2.2mM where N=3. N=7 for WNK1KD. b) Effects of increasing [Cl-] on kinase activity of the WNK3 kinase domain (WNK3KD) and the WNK4 kinase domain (WNK4KD) in vitro as in a. WNK4KD activity was inhibited at much lower [Cl-] than WNK3KD. N=5 for WNK3KD. WNK4KD data is the same as shown in a. c) Representative Western blots for pSPAK and total SPAK abundance from a WNK1KD kinase assay with increasing [Cl-] (mM). d) Representative Western blots for pSPAK and total SPAK abundance from a WNK3KD kinase assay with increasing [Cl-] (mM). e) Representative Western blots for pSPAK and total SPAK abundance from a WNK4KD kinase assay with increasing [Cl-] between 0 and 70 mM. f) Same as e, but for 0mM and 150mM [Cl-].
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814375&req=5

Figure 3: Effects of [Cl-] on in vitro WNK kinase activity using SPAK as a substratea) Effects of increasing [Cl-] on kinase activity on the WNK1 kinase domain (WNK1KD) and the WNK4 kinase domain (WNK4KD) in vitro. SPAK was used as the substrate. WNK4KD activity was inhibited at much lower [Cl-] than WNK1KD. For WNK4KD, N=4 for all data points except log[Cl-]=2.2mM where N=3. N=7 for WNK1KD. b) Effects of increasing [Cl-] on kinase activity of the WNK3 kinase domain (WNK3KD) and the WNK4 kinase domain (WNK4KD) in vitro as in a. WNK4KD activity was inhibited at much lower [Cl-] than WNK3KD. N=5 for WNK3KD. WNK4KD data is the same as shown in a. c) Representative Western blots for pSPAK and total SPAK abundance from a WNK1KD kinase assay with increasing [Cl-] (mM). d) Representative Western blots for pSPAK and total SPAK abundance from a WNK3KD kinase assay with increasing [Cl-] (mM). e) Representative Western blots for pSPAK and total SPAK abundance from a WNK4KD kinase assay with increasing [Cl-] between 0 and 70 mM. f) Same as e, but for 0mM and 150mM [Cl-].

Mentions: According to a model we previously developed, variations in extracellular [K+] such as those studied above would generate [Cl-]i concentrations in DCT cells of 10-20 mM. Given this, the data of BazĂșa-Valenti et al.22, and the proposed importance of WNK4 for this effect, we determined the Cl- sensitivity of WNK4, and compared it to that of WNK1 and WNK3. In vitro kinase assays were performed using the various WNK kinase domains (WNKKD) and SPAK as the physiologically-relevant kinase target. Our approach was similar to that reported by Piala and colleagues, except that they employed the generic substrate, myelin basic protein (MBP) for most of their studies.8 We used E. coli to produce the three WNKs, a system that has been shown to yield phosphorylated and active WNKs.8 Although Cl- inhibited all 3 WNKs, it inhibited WNK4KD kinase activity at much lower concentrations than it did WNK1KD or WNK3KD (Fig 3a, b). WNK4KD was inhibited in the physiological range for DCT [Cl-]i10, 24, 25 with the most potent effects between 0 and 40 mM (Fig 3e,f). WNK1KD and WNK3KD were inhibited at much higher levels, with WNK1KD being inhibited between 60 and 150 mM and WNK3KD inhibited between 100 and 150 mM (Fig 3c, d). Note, that a fraction of the GST fusion SPAK protein was truncated on its C-terminus during preparation (Supplemental Fig 1). Phosphorylation of both bands was quantified for these studies.


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 [Cl-] on in vitro WNK kinase activity using SPAK as a substratea) Effects of increasing [Cl-] on kinase activity on the WNK1 kinase domain (WNK1KD) and the WNK4 kinase domain (WNK4KD) in vitro. SPAK was used as the substrate. WNK4KD activity was inhibited at much lower [Cl-] than WNK1KD. For WNK4KD, N=4 for all data points except log[Cl-]=2.2mM where N=3. N=7 for WNK1KD. b) Effects of increasing [Cl-] on kinase activity of the WNK3 kinase domain (WNK3KD) and the WNK4 kinase domain (WNK4KD) in vitro as in a. WNK4KD activity was inhibited at much lower [Cl-] than WNK3KD. N=5 for WNK3KD. WNK4KD data is the same as shown in a. c) Representative Western blots for pSPAK and total SPAK abundance from a WNK1KD kinase assay with increasing [Cl-] (mM). d) Representative Western blots for pSPAK and total SPAK abundance from a WNK3KD kinase assay with increasing [Cl-] (mM). e) Representative Western blots for pSPAK and total SPAK abundance from a WNK4KD kinase assay with increasing [Cl-] between 0 and 70 mM. f) Same as e, but for 0mM and 150mM [Cl-].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814375&req=5

Figure 3: Effects of [Cl-] on in vitro WNK kinase activity using SPAK as a substratea) Effects of increasing [Cl-] on kinase activity on the WNK1 kinase domain (WNK1KD) and the WNK4 kinase domain (WNK4KD) in vitro. SPAK was used as the substrate. WNK4KD activity was inhibited at much lower [Cl-] than WNK1KD. For WNK4KD, N=4 for all data points except log[Cl-]=2.2mM where N=3. N=7 for WNK1KD. b) Effects of increasing [Cl-] on kinase activity of the WNK3 kinase domain (WNK3KD) and the WNK4 kinase domain (WNK4KD) in vitro as in a. WNK4KD activity was inhibited at much lower [Cl-] than WNK3KD. N=5 for WNK3KD. WNK4KD data is the same as shown in a. c) Representative Western blots for pSPAK and total SPAK abundance from a WNK1KD kinase assay with increasing [Cl-] (mM). d) Representative Western blots for pSPAK and total SPAK abundance from a WNK3KD kinase assay with increasing [Cl-] (mM). e) Representative Western blots for pSPAK and total SPAK abundance from a WNK4KD kinase assay with increasing [Cl-] between 0 and 70 mM. f) Same as e, but for 0mM and 150mM [Cl-].
Mentions: According to a model we previously developed, variations in extracellular [K+] such as those studied above would generate [Cl-]i concentrations in DCT cells of 10-20 mM. Given this, the data of BazĂșa-Valenti et al.22, and the proposed importance of WNK4 for this effect, we determined the Cl- sensitivity of WNK4, and compared it to that of WNK1 and WNK3. In vitro kinase assays were performed using the various WNK kinase domains (WNKKD) and SPAK as the physiologically-relevant kinase target. Our approach was similar to that reported by Piala and colleagues, except that they employed the generic substrate, myelin basic protein (MBP) for most of their studies.8 We used E. coli to produce the three WNKs, a system that has been shown to yield phosphorylated and active WNKs.8 Although Cl- inhibited all 3 WNKs, it inhibited WNK4KD kinase activity at much lower concentrations than it did WNK1KD or WNK3KD (Fig 3a, b). WNK4KD was inhibited in the physiological range for DCT [Cl-]i10, 24, 25 with the most potent effects between 0 and 40 mM (Fig 3e,f). WNK1KD and WNK3KD were inhibited at much higher levels, with WNK1KD being inhibited between 60 and 150 mM and WNK3KD inhibited between 100 and 150 mM (Fig 3c, d). Note, that a fraction of the GST fusion SPAK protein was truncated on its C-terminus during preparation (Supplemental Fig 1). Phosphorylation of both bands was quantified for these studies.

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