Newly discovered subunits of chloride transporters shed light on the mechanism of severe neurological diseases

Regulated transport of ions, such as sodium or chloride, across biological membranes is essential for the function of cells and the organism. Mutations in all nine genes of the CLC family of chloride transporters often lead to severe hereditary diseases in humans.

Function and disease relevance of CLC chloride channels

This gene family, discovered decades ago by Prof. Thomas Jentsch, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Max Delbrück Center for Molecular Medicine (MDC) & NeuroCure PI codes both for channels that form chloride-selective 'holes' in the outer cell membrane and for proteins that exchange chloride for protons (H+) via the membranes of intracellular vesicles. Mutations in CLC chloride channels lead, for example, to muscle stiffness or severe salt loss via the kidney, while mutations of intracellular chloride/proton exchangers can lead to protein loss into the urine and kidney stones (ClC-5), osteopetrosis (ClC-7), or neurological diseases (ClC-3 to ClC-7). This is probably due to a combination of altered acidification, electrical voltage, or chloride concentration of the corresponding vesicles (endosomes or lysosomes).  These parameters influence both the intracellular transport of the vesicle and its function, e.g. the degradation of proteins. However, the regulation of CLC transporters remained mysterious.

In recent years, the Jentsch research group, together with human geneticists, has been able to show that mutations in ClC-3 and ClC-4 sometimes lead to the most severe neurological diseases. However, biophysical analysis of the mutants often revealed no evidence of functional defects.

Schematic illustration of selected TMEM9 functions

TMEM9 and TMEM9B: Vital companions of the CLC transporters

In collaboration with Prof. Bernd Fakler, Freiburg, the research group has now discovered that two previously poorly characterized smaller proteins bind directly to ClC-3, -4, and -5 and represent so-called obligate beta subunits of these transporters. The FMP group discovered that these new subunits (TMEM9 and TMEM9B) have a diverse influence: On the one hand, they are necessary for the stability of the CLC transporters. This could be shown with KO mice in which the genes of the subunits were destroyed. The importance of the subunits is underlined by the fact that mice lacking both proteins die. On the other hand, both proteins also influence the localization of the CLC exchangers within the cell. The study by Prof. Thomas Jentsch and other scientists has now been published in the journal Nature Communications.

Perhaps most important was the surprising discovery that the new subunits almost completely block the ion transport of the CLC transporters. The working group suspected that disease-causing ClC-3 and ClC-4 mutations reduce this blockade. The group was able to demonstrate this with a newly developed test: Cellular overproduction of ClC-3 leads to swollen vesicles due to pathologically increased chloride uptake (image), which is prevented by simultaneous expression of TMEM9 subunits.  However, this blockade was absent in several pathogenic CLC mutants, which by themselves showed no effect. It thus became clear that these previously enigmatic mutants exhibit pathologically increased transport activity under normal conditions, i.e. in the presence of the beta subunits. The pharmacological inhibition of the transporters by drugs yet to be developed could be useful for the treatment of severe neurological symptoms in patients with specific mutations.

Targeted mutations showed that the C-terminus, the 'end' of the protein, inhibits ion transport. Together with the position of the patient mutations, a concrete model of interaction sites of the CLCs with the beta subunits could now be developed. This model was largely confirmed in collaboration with Prof. Richard Hite, New York, by creating a cryo-EM structure of the CLC/TMEM9 complex, which has not yet been finally published.

At the same time, the researchers were looking for clues as to how this inhibition can also be resolved a little physiologically. “Initial data suggest that this process takes place through phosphorylation, i.e. a chemical modification of the protein,” says Prof. Thomas Jentsch. “This is a very efficient way of regulating this channel.”

The next steps in the research will be to identify the proteins that phosphorylate the CLC or TMEM9 proteins and to investigate whether this process can be influenced pharmacologically.

Publication:
Rosa Planells-Cases, Viktoriia Vorobeva, Sumanta Kar, Franziska W. Schmitt, Uwe Schulte, Marina Schrecker, Richard K. Hite, Bernd Fakler, Thomas J. Jentsch. Endosomal chloride/proton exchangers need inhibitory TMEM9 ß-subunits for regulation and prevention of disease-causing overactivity.

DOI: https://doi.org/10.1038/s41467-025-58546-3

Source: Press release FMP

Contact: 
Prof. Dr. Dr. Thomas Jentsch
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Max Delbrück Center for Molecular Medicine (MDC)
+49 30 94793 521
jentsch@fmp-berlin.de