The erythroid K-Cl cotransport inhibitor [(dihydroindenyl)oxy]acetic acid blocks erythroid Ca²⁺-activated K⁺ channel KCNN4

Abstract

Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca2+-activated K+ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying expression and regulation of other K+ transporters and K+ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K+ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K+ uptake. In contrast, DIOA exhibited no inhibitory effect on K+ influx into A23187-treated red cells of Kcnn4-/- mice. DOIA inhibition of human KCa3.1 was validated (IC50 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.

Clinical implication of changes in serum cations and anions on clinical severity in sickle cell disease: A case–control study in a tertiary center

“…5 Other articles also proposed that such abnormality could result in end-organ damage with increased morbidity like kidney dysfunction, cerebrovascular stroke, and mortality. 4,5,18 The results of the present study, low serum sodium and chloride, agreed with the above findings (►Table 4) and propose that dehydration prevention would help reduce the crisis episodes. Besides serum electrolytes, serum calcium and magnesium were also grossly reduced in the cases of their healthy contemporary group (►Table 3).…”

Variant spectrum of PIEZO1 and KCNN4 in Japanese patients with dehydrated hereditary stomatocytosis

“…The novel p.A279T, first identified in our study, is located near p.V282M and p.V282E. The 231-289 th amino acids of KCNN4 form a two-pore potassium channel domain, and the 304-377 th amino acids form a calmodulin-binding domain 24,33 . These regions are highly conserved and have an important role in KCNN4.…”