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Metabolic energy sensing by mammalian CLC anion/proton exchangers.

ABSTRACT: CLC anion/proton exchangers control the pH and [Cl- ] of the endolysosomal system that is essential for cellular nutrient uptake. Here, we use heterologous expression and whole-cell electrophysiology to investigate the regulation of the CLC isoforms ClC-3, ClC-4, and ClC-5 by the adenylic system components ATP, ADP, and AMP. Our results show that cytosolic ATP and ADP but not AMP and Mg2+ -free ADP enhance CLC ion transport. Biophysical analysis reveals that adenine nucleotides alter the ratio between CLC ion transport and CLC gating charge and shift the CLC voltage-dependent activation. The latter effect is suppressed by blocking the intracellular entrance of the proton transport pathway. We suggest, therefore, that adenine nucleotides regulate the internal proton delivery into the CLC transporter machinery and alter the probability of CLC transporters to undergo silent non-transporting cycles. Our findings suggest that the CBS domains in mammalian CLC transporters serve as energy sensors that regulate vesicular Cl- /H+ exchange by detecting changes in the cytosolic ATP/ADP/AMP equilibrium. Such sensing mechanism links the endolysosomal activity to the cellular metabolic state.

SUBMITTER: Grieschat M

PROVIDER: S-EPMC7271328 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Metabolic energy sensing by mammalian CLC anion/proton exchangers.

Grieschat Matthias M Guzman Raul E RE Langschwager Katharina K Fahlke Christoph C Alekov Alexi K AK

EMBO reports 20200510 6

CLC anion/proton exchangers control the pH and [Cl<sup>-</sup> ] of the endolysosomal system that is essential for cellular nutrient uptake. Here, we use heterologous expression and whole-cell electrophysiology to investigate the regulation of the CLC isoforms ClC-3, ClC-4, and ClC-5 by the adenylic system components ATP, ADP, and AMP. Our results show that cytosolic ATP and ADP but not AMP and Mg<sup>2+</sup> -free ADP enhance CLC ion transport. Biophysical analysis reveals that adenine nucleot ...[more]

PMID: 32390228

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Project description:ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.

Project description:Virulent C. difficile toxins TcdA and TcdB invade host intestinal epithelia by endocytosis and use the acidic environment of intracellular vesicles for further processing and activation. We investigated the role of ClC-5, a chloride/proton exchanger expressed in the endosomes of gastrointestinal epithelial cells, in the activation and processing of C. difficile toxins. Enhanced intoxication by TcdA and TcdB was observed in cells expressing ClC-5 but not ClC-4, another chloride/proton exchanger with similar function but different localization. In accordance with the established physiological function of ClC-5, its expression lowered the endosomal pH in HEK293T cells by approximately 0.6 units and enhanced approximately 5-fold the internalization of TcdA. In colon HT29 cells, 34% of internalized TcdA localized to ClC-5-containing vesicles defined by colocalization with Rab5, Rab4a, and Rab7 as early and early-to-late of endosomes but not as Rab11-containing recycling endosomes. Impairing the cellular uptake of TcdA by deleting the toxin CROPs domain did not abolish the effects of ClC-5. In addition, the transport-incompetent mutant ClC-5 E268Q similarly enhanced both endosomal acidification and intoxication by TcdA but facilitated the internalization of the toxin to a lower extent. These data suggest that ClC-5 enhances the cytotoxic action of C. difficile toxins by accelerating the acidification and maturation of vesicles of the early and early-to-late endosomal system. The dispensable role of electrogenic ion transport suggests that the voltage-dependent nonlinear capacitances of mammalian CLC transporters serve important physiological functions. Our data shed light on the intersection between the endocytotic cascade of host epithelial cells and the internalization pathway of the large virulence C. difficile toxins. Identifying ClC-5 as a potential specific host ion transporter hijacked by toxins produced by pathogenic bacteria widens the horizon of possibilities for novel therapies of life-threatening gastrointestinal infections.

Dataset Information

Metabolic energy sensing by mammalian CLC anion/proton exchangers.

Publications

Metabolic energy sensing by mammalian CLC anion/proton exchangers.

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