Characterization of constitutive and acid-induced outwardly rectifying chloride currents in immortalized mouse distal tubular cells

Abstract

Thiazides block Na+ reabsorption while enhancing Ca2+ reabsorption in the kidney. As previously demonstrated in immortalized mouse distal convoluted tubule (MDCT) cells, chlorothiazide application induced a robust plasma membrane hyperpolarization, which increased Ca2+ uptake. This essential thiazide-induced hyperpolarization was prevented by the Cl− channel inhibitor 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), implicating NPPB-sensitive Cl− channels, however the nature of these Cl− channels has been rarely described in the literature. Here we show that MDCT cells express a dominant, outwardly rectifying Cl− current at extracellular pH 7.4. This constitutive Cl− current was more permeable to larger anions (Eisenman sequence I; I− > Br− ≥ Cl−) and was substantially inhibited by > 100 mM [Ca2+]o, which distinguished it from ClC-K2/barttin. Moreover, the constitutive Cl− current was blocked by NPPB, along with other Cl− channel inhibitors (4,4′-diisothiocyanatostilbene-2,2′-disulfonate, DIDS; flufenamic acid, FFA). Subjecting the MDCT cells to an acidic extracellular solution (pH < 5.5) induced a substantially larger outwardly rectifying NPPB-sensitive Cl− current. This acid-induced Cl− current was also anion permeable (I− > Br− > Cl−), but was distinguished from the constitutive Cl− current by its rectification characteristics, ion sensitivities, and response to FFA. In addition, we have identified similar outwardly rectifying and acid-sensitive currents in immortalized cells from the inner medullary collecting duct (mIMCD-3 cells). Expression of an acid-induced Cl− current would be particularly relevant in the acidic IMCD (pH < 5.5). To our knowledge, the properties of these Cl− currents are unique and provide the mechanisms to account for the Cl− efflux previously speculated to be present in MDCT cells.

Identification of TMEM206 proteins as pore of ASOR acid-sensitive chloride channels

“…The wide tissue expression pattern of TMEM206 indicated by public databases (Fig. 1E) agreed with the finding that I Cl,H has been found in every cell studied so far [11][12][13][14][15][16][18][19][20]. TMEM206 has orthologs in vertebrates and in the hemichordate Saccoglossus kowalevskii (acorn worm) ( Fig.…”

“…The observation of I Cl,H currents in every investigated mammalian cell type [11][12][13][14][15][16][18][19][20][21] suggests that ASOR may be expressed in all tissues. Such a wide expression pattern indicates that this channel has important physiological functions.…”

Acid- and Volume-Sensitive Chloride Currents in Human Chondrocytes

“…Acid-sensitive outwardly rectifying currents with remarkably similar pH sensitivities, phenotypes, activation kinetics, and pharmacological profiles have been described in various cell types like microglia, myocytes, HeLa cells, neurons, Sertoli cells, distal tubular cells, erythrocytes, or osteoclasts (Auzanneau et al, 2003;Nobles et al, 2004;Lambert and Oberwinkler, 2005;Wang et al, 2007;Kajiya et al, 2009;Sato-Numata et al, 2013Capurro et al, 2015;Valinsky et al, 2017;Kittl et al, 2019), but the knowledge on acid-sensitive Cl − currents in chondrocytes is sparse. There is only one report on an ASOR-type Cl − current in human OUMS-27 chondrocytes (Kurita et al, 2015).…”

“…In different cell types, extracellular acidification elicits an acidsensitive outwardly rectifying (ASOR) Cl − current (Auzanneau et al, 2003;Nobles et al, 2004;Lambert and Oberwinkler, 2005;Yamamoto and Ehara, 2006;Wang et al, 2007;Kajiya et al, 2009;Kucherenko et al, 2009;Sato-Numata et al, 2013Capurro et al, 2015;Kurita et al, 2015;Valinsky et al, 2017;Kittl et al, 2019). The ASOR current shows similarities to VSOR currents like its pharmacological profile, ion permeability sequence, and outward rectification.…”

Aldosterone, SGK1, and ion channels in the kidney

“…The relationship between aldosterone, SGK1, and Mg 2+ permeable channels represents a largely unexplored field of renal electrolyte regulation. While many Mg 2+ permeable channels have been identified in DCT primary cells and cell lines, such as transient receptor potential melastatin (TRPM)6 [ 87 – 89 ], TRPM7 [ 89 – 91 ], MagT1 [ 92 , 93 ], and ACDP2/CNNM2 [ 94 ], few have been studied with aldosterone. TRPM6 [ 87 , 95 ] and TRPM7 [ 91 , 96 – 98 ] are further complex, as they comprise Mg 2+ permeable, nonselective cation channels fused to a C-terminal α-kinase domain.…”