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

https://doi.org/10.1016/j.bbagen.2017.05.004Get rights and content
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Highlights

  • MDCT cells express a dominant NPPB-sensitive Cl current at pH 7.4.

  • The constitutive Cl current (pH 7.4) does not arise from ClC-K2/barttin.

  • MDCT cells also express an acid-induced NPPB-sensitive Cl current (pH < 5.5).

  • Both the constitutive and acid-induced Cl currents are unique.

  • mIMCD-3 cells express currents with similar biophysical properties.

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.

Abbreviations

2-APB
2-aminoethoxydiphenyl borate
DCT
distal convoluted tubule
DIDS
4,4′-diisothiocyanatostilbene-2,2′-disulfonate
FFA
flufenamic acid
HBE
human bronchial epithelial
HEK
human embryonic kidney
I-V
current-voltage
VLJP
liquid junction potential
MDCT
mouse distal convoluted tubule cell line
mIMCD-3
mouse inner medullary collecting duct cell line
NCC
Na+/Cl co-transporter
NPPB
5-Nitro-2-(3-phenylpropylamino) benzoic acid
TRPM
transient receptor potential melastatin
VRAC
volume-regulated anion channel

Keywords

Chloride current
Acid-induced chloride current
Acid-sensitive outwardly rectifying (ASOR) anion channel
Distal convoluted tubule
MDCT cells
mIMCD-3 cells

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