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ClC-K2 Cl^- channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.

ABSTRACT: The collecting duct is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Principal and intercalated cells perform different physiological tasks and exhibit distinctive morphology. However, acid-secreting A- and base secreting B-type of intercalated cells cannot be easily separated in functional studies. We used BCECF-sensitive intracellular pH (pH_i ) measurements in split-opened collecting ducts followed by immunofluorescent microscopy in WT and intercalated cell-specific ClC-K2^-/- mice to demonstrate that ClC-K2 inhibition enables to distinguish signals from A- and B-intercalated cells. We show that ClC-K2 Cl^- channel is expressed on the basolateral side of intercalated cells, where it governs Cl^- -dependent H⁺ /HCO₃^- transport. ClC-K2 blocker, NPPB, caused acidification or alkalization in different subpopulations of intercalated cells in WT but not ClC-K2^-/- mice. Immunofluorescent assessment of the same collecting ducts revealed that NPPB increased pH_i in AE1-positive A-type and decreased pH_i in pendrin-positive B-type of intercalated cells. Induction of metabolic acidosis led to a significantly augmented abundance and H⁺ secretion in A-type and decreased proton transport in B-type of intercalated cells, whereas metabolic alkalosis caused the opposite changes in intercalated cell function, but did not substantially change their relative abundance. Overall, we show that inhibition of ClC-K2 can be employed to discriminate between A- and B-type of intercalated cells in split-opened collecting duct preparations. We further demonstrate that this method can be used to independently monitor changes in the functional status and abundance of A- and B-type in response to systemic acid/base stimuli.

SUBMITTER: Pyrshev K

PROVIDER: S-EPMC9014849 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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ClC-K2 Cl<sup>-</sup> channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.

Pyrshev Kyrylo K Khayyat Naghmeh Hassanzadeh NH Stavniichuk Anna A Tomilin Viktor N VN Zaika Oleg O Ramkumar Nirupama N Pochynyuk Oleh O

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20220501 5

The collecting duct is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Principal and intercalated cells perform different physiological tasks and exhibit distinctive morphology. However, acid-secreting A- and base secreting B-type of intercalated cells cannot be easily separated in functional studies. We used BCECF-sensitive intracellular pH (pH<sub>i</sub> ) measurements in split-opened collecting ducts followed by immunofluorescent micro ...[more]

PMID: 35349181

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Project description:ClC-6 and ClC-7 are closely related, intracellular Cl- /H+ antiporters belonging to the CLC family of channels and transporters. They localize to acidic late endosomes and lysosomes and probably function in ionic homeostasis of these contiguous compartments. ClC-7 transport function requires association with the accessory protein Ostm1, whereas ClC-6 transport does not. To elucidate their roles in endo-lysosomes, we measured Cl- - and pH-dependences of over-expressed wild-type ClC-6 and ClC-7, as well as disease-associated mutants, using high-resolution recording protocols. Lowering extracellular Cl- (corresponding to luminal Cl- in endo-lysosomes) reduced ClC-6 currents, whereas it increased transport activity of ClC-7/Ostm1. Low extracellular Cl- activated ClC-7/Ostm 1 under acidic extracellular conditions, as well as under conditions of low intracellular chloride. Activation is conserved in ClC-7Y713C , a variant displaying disrupted PI(3,5)P2 inhibition. Detailed biophysical analysis of disease-associated ClC-6 and ClC-7 gain-of-function (GoF) variants, ClC-6Y553C and ClC-7Y713C , and the ClC-7Y577C and ClC-6Y781C correlates, identified additional functional nuances distinguishing ClC-6 and ClC-7. ClC-7Y577C recapitulated GoF produced by ClC-6Y553C . ClC-6Y781C displayed transport activation qualitatively similar to ClC-7Y713C , although current density did not differ from that of wild-type ClC-6. Finally, rClC-7R760Q , homologous to hClC-7R762Q , an osteopetrosis variant with fast gating kinetics, appeared indifferent to extracellular Cl- , identifying altered Cl- sensitivity as a plausible mechanism underlying disease. Collectively, the present studies underscore the distinct roles of ClC-6 and ClC-7 within the context of their respective localization to late endosomes and lysosomes. In particular, we suggest the atypical inhibition of ClC-7 by luminal Cl- serves to limit excessive intraluminal Cl- accumulation. KEY POINTS: ClC-6 and ClC-7 are late endosomal and lysosomal 2 Cl- /1 H+ exchangers, respectively. When targeted to the plasma membrane, both activate slowly at positive voltages. ClC-6 activity is decreased in low extracellular (i.e. luminal) chloride, whereas ClC-7 is activated by low luminal chloride, even at acidic pH. The functional gain-of-function phenotypes of the ClC-6 and ClC-7 disease mutations ClC-6Y553C and ClC-7Y715C are maintained when introduced in their respective homologues, ClC-7Y577C and ClC-6Y781C , with all mutations retaining chloride dependence of the respective wild type (WT). An osteopetrosis mutation of ClC-7 displaying fast gating kinetics (R762Q) was less sensitive to extracellular chloride compared to WT. The opposing substrate dependences of ClC-6 and ClC-7 Cl- / H+ exchangers point to non-overlapping physiological functions, leading us to propose that inhibition of ClC-7 by luminal chloride and protons serves to prevent osmotic stress imposed by hyper-accumulation of chloride.

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Dataset Information

ClC-K2 Cl^- channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.

Publications

ClC-K2 Cl<sup>-</sup> channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.

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Dataset Information

ClC-K2 Cl- channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.

Publications

ClC-K2 Cl<sup>-</sup> channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

ClC-K2 Cl^- channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.