Wen D, Cornelius RJ, Yuan Y, Sansom SC. Regulation of BK-␣ expression in the distal nephron by aldosterone and urine pH.

Increased flow in the distal nephron induces K secretion through the large-conductance, calcium-activated K channel (BK), which is primarily expressed in intercalated cells (IC). Since flow also increases ATP release from IC, we hypothesized that purinergic signaling has a role in shear stress (τ; 10 dynes/cm(2)) -induced, BK-dependent, K efflux. We found that 10 μM ATP led to increased IC Ca concentration, which was significantly reduced in the presence of the P(2) receptor blocker suramin or calcium-free buffer. ATP also produced BK-dependent K efflux, and IC volume decrease. Suramin inhibited τ-induced K efflux, suggesting that K efflux is at least partially dependent on purinergic signaling. BK-β4 small interfering (si) RNA, but not nontarget siRNA, decreased ATP secretion and both ATP-dependent and τ-induced K efflux. Similarly, carbenoxolone (25 μM), which blocks connexins, putative ATP pathways, blocked τ-induced K efflux and ATP secretion. Compared with BK-β4(-/-) mice, wild-type mice with high distal flows exhibited significantly more urinary ATP excretion. These data demonstrate coupled electrochemical efflux between K and ATP as part of the mechanism for τ-induced ATP release in IC.

The large conductance, calcium-activated BK-α/β4 potassium channel, localized to the intercalated cells of the distal nephron, mediates potassium secretion during high potassium, alkaline diets. Here we determine whether BK-α/β4-mediated potassium transport is dependent on epithelial sodium channel (ENaC)-mediated sodium reabsorption. We maximized sodium-potassium exchange in the distal nephron by feeding mice a low sodium, high potassium diet. Wild type and BK-β4 knockout mice were maintained on low sodium, high potassium, alkaline diet or a low sodium, high potassium, acidic diet for 7–10 days. Wild type mice maintained potassium homeostasis on the alkaline but not acid diet. BK-β4 knockout mice could not maintain potassium homeostasis on either diet. During the last 12 hours of diet, wild type mice on either a regular, alkaline or an acid diet, or knockout mice on an alkaline diet were administered amiloride (an ENaC inhibitor). Amiloride enhanced sodium excretion in all wild type and knockout groups to similar values; however, amiloride diminished potassium excretion by 59% in wild type but only by 33% in knockout mice on an alkaline diet. Similarly, amiloride decreased the transtubular potassium gradient by 68% in wild type but only by 42% in knockout mice on an alkaline diet. Amiloride treatment equally enhanced sodium excretion and diminished potassium secretion in knockout mice on an alkaline diet and wild type mice on an acid diet. Thus, the enhanced effect of amiloride on potassium secretion in wild type compared to knockout mice on the alkaline diet, clarify a BK- α/β4-mediated potassium secretory pathway in intercalated cells driven by ENaC-mediated sodium reabsorption linked to bicarbonate secretion.

Familial hyperkalemic hypertension is caused by mutations in with-no-lysine kinases (WNKs) or in proteins that mediate their degradation, kelch-like 3 (KLHL3) and cullin 3 (CUL3). Although the mechanisms by which WNK and KLHL3 mutations cause the disease are now clear, the effects of the disease-causing CUL3Δ403-459 mutation remain controversial. Possible mechanisms, including hyperneddylation, altered ubiquitin ligase activity, decreased association with the COP9 signalosome (CSN), and increased association with and degradation of KLHL3 have all been postulated. Here, we systematically evaluated the effects of Cul3Δ403-459 using cultured kidney cells. We first identified that the catalytically active CSN subunit jun activation domain-binding protein-1 (JAB1) does not associate with the deleted Cul3 4-helix bundle domain but instead with the adjacent α/β domain, suggesting that altered protein folding underlies the impaired binding. Inhibition of deneddylation with JAB1 siRNA increased Cul3 neddylation and decreased KLHL3 abundance, similar to the Cul3 mutant. We next determined that KLHL3 degradation has both ubiquitin ligase-dependent and -independent components. Proteasomal KLHL3 degradation was enhanced by Cul3Δ403-459; however, autophagic degradation was also upregulated by this Cul3 mutant. Finally, to evaluate whether deficient substrate adaptor was responsible for the disease, we restored KLHL3 to wild-type (WT) Cul3 levels. In the absence of WT Cul3, WNK4 was not degraded, demonstrating that Cul3Δ403-459 itself cannot degrade WNK4; conversely, when WT Cul3 was present, as in diseased humans, WNK4 degradation was restored. In conclusion, deletion of exon 9 from Cul3 generates a protein that is itself ubiquitin-ligase defective but also capable of enhanced autophagocytic KLHL3 degradation, thereby exerting dominant-negative effects on the WT allele.