Managing the chemical workplaces of the cell

Our cells contain many small compartments in which specialized molecules perform their jobs. Membranes around the compartments keep the molecules from escaping and help provide special chemical environments that they need to work. Lysosomes, for example, have high levels of acid in which powerful enzymes break down debris and foreign matter. Creating and maintaining this environment is the job of proteins in the membranes that surround the compartments, and defects in the process can lead to serious diseases. Now the lab of Thomas Jentsch at the MDC and FMP has revised our view of how some of the proteins function, which will likely provide important insights into the diseases. Their work is reported in two studies that appeared in the June 11 issue of Science.

Creating an acid environment in compartments such as endosomes or lysosomes is the job of a proton pump (green). The rising positive charge has to be compensated for; in the past researchers thought that chloride channels (purple) were responsible (a). Thomas' lab has discovered that ClC-5 and ClC-7 are not channels in endosomes and lysosomes, but instead are chloride-proton exchangers (b). The lab developed strains of mice in which the exchangers are converted to channels (c). These mice had phenotypes similar to knock-out mice, suggesting that chloride, which is accumulated by the exchangers, has novel, crucial roles in these cellular compartments. 

To create higher levels of acid than are found in the rest of the cell, protons are snatched from outside the compartment and pumped inside. Because protons have a positive electrical charge, this would result in a positive voltage inside the compartment, which would very soon inhibit further pumping. Acidification wouldn't be possible without a compensation for this charge. Over the past several decades, it has been thought that the compensation was achieved by a passive uptake of negatively charged chloride ions. In this "classical textbook" model, chloride was thought to flow through chloride "channels" – proteins in the membrane with minute holes through which chloride can pass.  

Members of the family of molecules called CLC anion transporters, which was discovered by Thomas' lab, were believed to act as such chloride channels in lysosomes and other cellular compartments. Thomas’ lab had shown that lysosome membranes contain a member of this protein family called ClC-7 which plays a very important role: its loss in mice or in humans led to defects in lysosomes, neurodegeneration, and an excessive mineralization of bone called osteopetrosis.

"The traditional view has been that ClC-7 helps manage levels of acid in the lysosome," Thomas says. "But we've found evidence that it is actually doing something different. This is crucial to our understanding of lysosomal storage diseases, in which cells can not efficiently degrade waste material. This material then accumulates within cells, which may eventually lead to their death, as in the case of neurodegeneration."

If ClC-7 were regulating the amount of acid in the compartments, then removing the protein ought to cause a change in the acidity of the lysosome. But an experiment carried out by Thomas' lab a few years ago revealed that this wasn't the case. The scientists developed a strain of mouse without ClC-7. Although the loss of the molecule didn't change levels of acid in the lysosomes, the animals still developed the symptoms of lysosomal storage diseases. Thomas and his colleagues speculated that ClC-7 might have another important function.

They had a hint about what it might be from previous work on another member of the chloride channel family called ClC-5, found in compartments called endosomes in kidney cells. Endosomes transport proteins that have been taken up at the cell surface to lysosomes, which serve as cellular trash bins. Defects in ClC-5 cause the malfunction of the endosomal compartment and lead to Dent's disease, in which cells don't take up wastes properly; instead, proteins are lost into the urine and patients develop kidney stones.

The Jentsch lab had shown that ClC-5 doesn't serve as a passive channel for chloride diffusion. Instead, it strictly couples chloride transport to a countertransport of protons, making ClC-5 an exchanger rather than a channel. Because it exchanges negative for positive charges, the process efficiently generates electrical current which can compensate for the current from the proton pump. The lab showed that endosomes don't acquire their normal levels of acid when ClC-5 is lost. However, why should nature have chosen such a complicated exchange process, if a simple chloride channel – as the classical model suggested – could also do the trick?

If one could convert these exchangers into pure chloride channels, the scientists believed, they ought to still carry out their functions in compensating for the rise in charge. If mice with channels – but not exchangers – still developed disease, it wouldn't be due to changes in levels of acid; instead, the problem would be that the acquisition of protons had become uncoupled from the import of chloride. In the two new studies, headed by postdocs Stefanie Weinert and Gaia Novarino, the scientists converted the lysosomal ClC-7 and the endosomal ClC-5 into "classic" channels. This was possible with only a minute change in the protein: altering only one of the 800 amino acids that made up each of the molecules.

"The results were very surprising," Thomas says. “The alteration in ClC-5 led to mice which developed a phenotype that is the same as you find if they are totally lacking the protein. Converting ClC-7 to a ‘channel’ produced the same severe neurodegeneration as in the knock-out mouse, with somewhat less severe osteopetrosis. This demonstrates that the cause of the diseases is not a change in acid levels in the compartments, as most people have thought. We think it's due to something else: it means that these proteins normally use the downflow of protons, which are more concentrated in these acidic compartments than in the rest of the cell, to drive the import of chloride. If ClC-5 and ClC-7 don't function properly, there isn't enough chloride in the compartment. And that is clearly important, because it leads to disease."

Demonstrating that this was the case required the development and improvement of methods to measure chloride concentrations and levels of acid in lysosomes and endosomes. They also needed new mathematical models to interpret changes in the concentration of ions and the behavior of the membranes.

"This has important implications for our understanding of the operation of these cellular compartments and likely many others," Thomas says. "It suggests that instead of focusing only on changes in acidification as the cause of disease, we need to understand the role of the accumulation of chloride ions in those compartments . That's a fundamental change in perspective."

- Russ Hodge

Highlight Reference:

Novarino G, Weinert S, Rickheit G, Jentsch TJ. Endosomal chloride-proton exchange rather than chloride conductance is crucial for renal endocytosis. Science. 2010 Jun 11;328(5984):1398-401. Epub 2010 Apr 29.
Weinert S, Jabs S, Supanchart C, Schweizer M, Gimber N, Richter M, Rademann J, Stauber T, Kornak U, Jentsch TJ. Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal Cl- accumulation. Science. 2010 Jun 11;328(5984):1401-3.

Full text of the paper by Novarino et al.
Full text of the paper by Weinert et al.
2010 Research report of the Jentsch lab (page 154)