Final Report Summary - CYTOVOLION (Ion homeostasis and volume regulation of cells and organelles)
When cells are exposed to external solutions that contain less salt, they will swell – a dangerous situation as the cells may even burst and die at some point. Therefore, they must have mechanisms to reduce their volume, a process called regulatory volume decrease. Cell volume regulation is also important during cell division, growth, and programmed cell death. It has been suspected that a certain ion channel, called VRAC, is crucially involved in these processes. Ion channels are proteins that reside in the plasma membrane, the layer of lipids that enclose cells, and form regulated pores through which substances like ions (components of salt) can pass. The biophysical properties of VRAC channels have been known for almost 30 years, but in spite of many attempts by many groups, the underlying genes and proteins have remained unknown. However, the identification of the molecular composition of VRAC is essential for understanding its regulation and role in health and disease.
The most important goal of our project was therefore the identification of VRAC and the subsequent characterization of some of its roles. Using a very broad screen, in which we suppressed the function of each of the ~20,000 human genes singly and examined the impact on ion transport, we achieved the breakthrough. We identified a protein, named LRRC8A, as an essential component of VRAC. We found that it needs other members of the same gene family (LRRC8B-E) to form functional VRAC channels. Our subsequent experiments showed that VRAC also conducts small organic molecules like amino acids, neurotransmitters, and even clinically important drugs. VRAC-mediated release of signaling molecules from cells may be important for data processing in the brain and for pathological conditions such as stroke, whereas VRAC-mediated drug uptake influences, for example the response of tumors to drugs as we have shown for the widely used anti-cancer drug cisplatin. The transport of these substances depends on the presence of specific LRRC8 proteins in the channel complex, a finding that may potentially allow specific pharmacological interventions in disease. Analysis of the effect of mutations in LRRC8 proteins have already provided us with insights into the molecular working of VRAC. The identification of proteins constituting VRAC has swung the door wide open to investigate its regulation, physiological and pathological roles – issues we are investigating using, for example, genetically modified mice.
Other important goals were the identification of mechanisms involved in ion homeostasis in intracellular vesicles. These are small compartments within the cell which are also enclosed by a lipid membrane. Formation of such vesicles can occur via formation from the outer (plasma) membrane (named endocytosis). These newly formed vesicles can take up from the exterior molecules that are destined for degradation in lysosomes (another intracellular vesicle that can be regarded as cellular trash bin). Interfering with the function of endosomes and lysosomes leads to a large variety of human diseases. The function of endosomes and lysosomes depends on the transport of ions across their membranes, and in the past we have shown that human mutations in several of these ion transport proteins lead to disease. In the current project, we have focused mainly on one of these transporters, ClC-7, and have shown that different aspects of ClC-7 (like ion exchange, electrical current, or interaction with other proteins) contribute differentially to the respective disease symptoms (thick fragile bones and brain pathology). Moreover, we examined the regulation of ClC-7 by the applied voltage, and studies to determine the ion concentrations in those compartments and their functional consequences are ongoing.
The most important goal of our project was therefore the identification of VRAC and the subsequent characterization of some of its roles. Using a very broad screen, in which we suppressed the function of each of the ~20,000 human genes singly and examined the impact on ion transport, we achieved the breakthrough. We identified a protein, named LRRC8A, as an essential component of VRAC. We found that it needs other members of the same gene family (LRRC8B-E) to form functional VRAC channels. Our subsequent experiments showed that VRAC also conducts small organic molecules like amino acids, neurotransmitters, and even clinically important drugs. VRAC-mediated release of signaling molecules from cells may be important for data processing in the brain and for pathological conditions such as stroke, whereas VRAC-mediated drug uptake influences, for example the response of tumors to drugs as we have shown for the widely used anti-cancer drug cisplatin. The transport of these substances depends on the presence of specific LRRC8 proteins in the channel complex, a finding that may potentially allow specific pharmacological interventions in disease. Analysis of the effect of mutations in LRRC8 proteins have already provided us with insights into the molecular working of VRAC. The identification of proteins constituting VRAC has swung the door wide open to investigate its regulation, physiological and pathological roles – issues we are investigating using, for example, genetically modified mice.
Other important goals were the identification of mechanisms involved in ion homeostasis in intracellular vesicles. These are small compartments within the cell which are also enclosed by a lipid membrane. Formation of such vesicles can occur via formation from the outer (plasma) membrane (named endocytosis). These newly formed vesicles can take up from the exterior molecules that are destined for degradation in lysosomes (another intracellular vesicle that can be regarded as cellular trash bin). Interfering with the function of endosomes and lysosomes leads to a large variety of human diseases. The function of endosomes and lysosomes depends on the transport of ions across their membranes, and in the past we have shown that human mutations in several of these ion transport proteins lead to disease. In the current project, we have focused mainly on one of these transporters, ClC-7, and have shown that different aspects of ClC-7 (like ion exchange, electrical current, or interaction with other proteins) contribute differentially to the respective disease symptoms (thick fragile bones and brain pathology). Moreover, we examined the regulation of ClC-7 by the applied voltage, and studies to determine the ion concentrations in those compartments and their functional consequences are ongoing.