Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K

Abstract

A potassium (K⁺ ) rich diet is known to have an antihypertensive effect that has been embodied by the NHLBI in the DASH diet. However, the molecular basis for this blood pressure-lowering effect has been unclear, until a recent study proposed a model in which the DCT cells of the kidney regulate their salt transport in response to variations in intracellular chloride ([Cl^- ]_i ), which are directly regulated by serum K⁺ . With the knowledge that WNK proteins are Cl^- sensors, and are a part of the WNK/SPAK/NCC signaling cascade which regulates the NCC, the main salt transporter in the distal nephron, we examined the effect of serum K⁺ on the ([Cl^- ]_i ) and, in turn its effect on the WNK4 signaling pathway in a "modified HEK 293T" cell line. Using a fluorescence-based approach in this cell line, we have shown that the membrane potential of the cell membrane is sensitive to the small changes in external KCl within the physiological range (2-5 mM), thus functioning as a K⁺ electrode. When the extracellular K⁺ was progressively increased (2-5 mM), the membrane depolarization lead to a subsequent increase in [Cl^- ]_i measured by fluorescence quenching of an intracellular chloride sensor. Increase in extracellular [K] resulted in a decrease in the phosphorylation of the WNK4 protein and its downstream targets, SPAK and NCC. This confirms that small changes in serum K can affect WNK4/SPAK/NCC signaling and transcellular Na⁺ flux through the DCT and provide a possible mechanism by which a K-rich DASH diet could reduce blood pressure.

Keywords: NCC; SPAK kinase; WNK kinases; chloride sensing domain; potassium.

Figure 1

HEK cells transfected with VSFP3.1 and KCNJ10 were exposed to different [KCl]_o. Fluorescence at 2 mM KCl recorded from 1 to 200 s was adjusted to a baseline of 100%. For those wells where [KCl]_o was changed, a baseline was obtained for the first 50 s in 2 mM KCl, after which the [KCl]_o was increased from 2 through 5 mM KCl in 0.5 mM steps, respectively (a). CFP quenching at different [KCl]_o. (b) Normalized response to KCl (ΔF/Fo) plotted semilogarithmically against final [K]o. Data points are means of triplicates. (c) Effect of K channel blockers on CFP quenching

Figure 2

HEK cells transfected with EYFP H148Q/I152L, KCNJ10, Clc‐kb, and barttin and exposed to different [KCl]_o. Fluorescence at 2 mM KCl recorded from 1 to 200 s was adjusted to a baseline of 100%. For those wells where [KCl]_o was changed, a baseline was obtained for the first 50 s in 2 mM KCl, after which the [KCl]_o was increased from 2 mM through 5 mM KCl, respectively. (a) EYFP quenching at different [KCl]_o. (b) Normalized response to KCl (ΔF/Fo) plotted against final [Ko]. Data points are means of four different experiments. (c) Effect of different channel blockers, such as Ba²⁺ (1 mM), Tertiapin Q (100 nM), Valsartan (100 µM), DIDS (200 µM) on EYFP quenching

Figure 3

Effect of [K]_o on WNK4 phosphorylation in HEK 293 T cells transfected with NCC alone or different forms of WNK4: WNK4 WT, WNK4 LLFF, and WNK4 LLFF KD, respectively. (a) Total protein lysates were blotted from HEK 293 T cells transfected with NCC and the WT WNK4 at four different external KCl. Rabbit phospho WNK4 Ser 575 antibody detected a 150 KDa WNK4 band. ß‐actin was the loading control. This is representative of Western blots replicated three times with cell lysates from three different passage numbers. (b) Quantification of Western blots showing phospho WNK4 levels normalized to its ß‐actin loading control. Error bars represent mean ± SEM of Western blots (n = 3). *p < .0001 by two‐tailed t test. (c) The effect of K channel blocker, Tertiapin Q on phospho WNK4 levels at 4 mM, and 5 mM KCl, respectively. (d) Phospho WNK4 levels in cells transfected with the three different WNK4 forms, and at four, different [K]o

Figure 4

Effect of [K]o on NCC phosphorylation in HEK 293 T cells transfected with NCC alone or with WNK4 WT. The transfected cells were treated with external KCl concentrations of 2, 3, 4, and 5 mM respectively. Cells were biotinylated. (a) Nonbiotinylated fraction of the cell lysates were blotted. Sheep pSPAK t373 antibody detected an approximately 60 KD band. (b) Quantification of Western blots showing phospho pSPAK levels normalized to its ß‐actin loading control. Error bars represent mean ± SEM of Western blots (n = 3). p < .05 by one‐way ANOVA. (c) Biotinylated protein lysates were blotted and rabbit pNCC Thr 58 antibody detected a 150 KDa phospho NCC band. (d) Quantification of Western blots showing biotinylated phospho NCC levels. Error bars represent mean ± SEM of Western blots (n = 3). *p < .05 by two‐tailed t test