P2Y receptor regulation of K2P channels that facilitate K⁺ secretion by human mammary epithelial cells

Abstract

The objective of this study was to determine the molecular identity of ion channels involved in K secretion by the mammary epithelium and to examine their regulation by purinoceptor agonists. Apical membrane voltage-clamp experiments were performed on human mammary epithelial cells where the basolateral membrane was exposed to the pore-forming antibiotic amphotericin B dissolved in a solution with intracellular-like ionic composition. Addition of the Na channel inhibitor benzamil reduced the basal current, consistent with inhibition of Na uptake across the apical membrane, whereas the K3.1 channel blocker TRAM-34 produced an increase in current resulting from inhibition of basal K efflux. Treatment with two-pore potassium (K2P) channel blockers quinidine, bupivacaine and a selective TASK1/TASK3 inhibitor (PK-THPP) all produced concentration-dependent inhibition of apical K efflux. qRT-PCR experiments detected mRNA expression for nine K2P channel subtypes. Western blot analysis of biotinylated apical membranes and confocal immunocytochemistry revealed that at least five K2P subtypes (TWIK1, TREK1, TREK2, TASK1, and TASK3) are expressed in the apical membrane. Apical UTP also increased the current, but pretreatment with the PKC inhibitor GF109203X blocked the response. Similarly, direct activation of PKC with phorbol 12-myristate 13-acetate produced a similar increase in current as observed with UTP. These results support the conclusion that the basal level of K secretion involves constitutive activity of apical K3.1 channels and multiple K2P channel subtypes. Apical UTP evoked a transient increase in K3.1 channel activity, but over time caused persistent inhibition of K2P channel function leading to an overall decrease in K secretion.

Gain-of-function mutations in KCNK3 cause a developmental disorder with sleep apnea

“…To reflect the heterozygous probands, we coexpressed equal amounts of WT and mutant TASK-1 mRNAs and measured their inhibition by either carbachol via endogenous muscarinic receptors or ATP via a coexpressed P2Y receptor 44 , 45 . We found that GPCR-mediated inhibition by either approach was markedly impaired in all DDSA mutant homomeric channels (Supplementary Fig.…”

Defective X-gating caused byde novogain-of-function mutations inKCNK3underlies a developmental disorder with sleep apnea

“…To reflect the heterozygous probands, we co-expressed equal amounts of WT and mutant TASK-1 mRNAs and measured their inhibition by either carbachol via endogenous muscarinic receptors, or by ATP via a coexpressed P2Y receptor as reported previously 41,42 . However, unlike WT TASK-1, we found that GPCR-mediated inhibition by either approach was markedly impaired in all 'heterozygous' DDSA mutant co-expressions (Fig.…”

Deficiency of the Two-Pore Potassium Channel KCNK9 Impairs Intestinal Epithelial Cell Survival and Aggravates Dextran Sodium Sulfate-Induced Colitis

“…To validate the detrimental effects caused by genetic knockout in Kcnk9 -/mice, we specifically blocked KCNK9 using the recently described substance [4,3-d]pyrimidin-4-yl]-4-piperidinyl]-1-butanone (PK-THPP) in wildtype and knockout animals and again acutely induced colitis with DSS treatment. 14,15 Pharmacologic inhibition of KCNK9 severely affected wild-type animals as indicated by weight loss (Figure 2A) and disease activity index (Figure 2B), but did not further modify the disease course of knockout animals. Barrier disruption also was more substantial in PK-THPP-treated wild-type animals (Figure 2E).…”

“…On days 6-9, mice received normal drinking water. One experimental group received PK-THPP, a potent antagonist of KCNK9, 14,15 which was administered intraperitoneally once daily throughout the experiment (10 mg/g body weight in 200 mL 1% dimethyl sulfoxide [DMSO]/40% polyethylene glycol 400/phosphatebuffered saline [PBS]) (DMSO; polyethylene glycol 400) Q22 , Sigma Aldrich, St. Louis, MO). Control animals received 200 mL 1% DMSO/40% polyethylene glycol 400/PBS intraperitoneally.…”