Mouse bulbus olfactorius under the microscope

Jentsch Lab

Physiology and Pathology of Ion Transport

Volume-stimulated organic osmolyte / anion channel VSOAC/VRAC

Research Project 2

Cells need to regulate their volume in face of osmotic stress and during cell growth, migration, and apoptosis. A key player in regulatory volume decrease is the volume-regulated anion channel VRAC (also known as VSOR or VSOAC) that opens upon cell swelling. Its opening leads to a passive efflux of chloride that is electrically balanced by K+-efflux through constitutively open K+ channels. Cell swelling also leads to an efflux of organic osmolytes like taurine or glutamate, but it was controversial whether this occurs through the same channel. As water transport across cellular membranes is driven by osmotic gradients, opening of VRAC/VSOAC leads to water efflux and to cell shrinkage (Figure 1).

Figure 1. Schematic diagram of VRAC function. VRAC is closed under resting conditions (top left) and opens after osmotic swelling. The resulting efflux of chloride and organic osmolytes decreases cellular osmolarity, resulting in water efflux and regulatory volume decrease.

Whereas the biophysical footprint of VRAC has been known since the late 1980s, and while the channel has been described and analyzed in hundreds of publications, attempts to identify the underlying molecule(s) have failed repeatedly. In collaboration with the in-house FMP screening facility, we recently used a genome-wide RNA interference screen to identify LRRC8A as an essential component of VRAC [20]. LRRC8A displays four transmembrane domains and a large cytoplasmic tail containing many leucine-rich repeats (LRRs). It has four close homologs (LRRC8B-E) and recent bioinformatic analysis had shown that LRRC8 proteins are distantly related to pannexins that form hexameric plasma membrane channels. Co-immunoprecipitation and intracellular trafficking experiments showed that LRRC8A can form heteromers with these other members. Using the novel CISPR-Cas genomic editing tool, we disrupted all five LRRC8 genes in cells singly and in several combinations (including a cell line with disruption of all five LRRC8 genes) and reconstituted selected LRRC8 isoforms by transfection [20]. Electrophysiological analysis revealed that only LRRC8A is essential for VRAC function, but that it needs at least one other LRRC8 isoform (LRRC8B-E) to yield currents. Different combinations of LRRC8A with other isoforms (e.g. LRRC8C or LRRC8E) yielded VRAC currents that differed in their inactivation kinetics, proving that LRRC8 proteins are integral parts of the channel and not just involved in its activation. As LRRC8 isoforms display differential tissue distribution our results also explain the differences in inactivation kinetics of VRAC currents in different tissues that had been enigmatic.

Importantly, we also showed that the efflux of the sulfoamino-acid taurine, an important cellular osmolyte, also depends on LRRC8 heteromers, showing that VRAC is indeed identical to VSOAC [20]. We suspect that the same channel conducts a number of other organic substances, several of which may be involved in signal transduction (e.g. taurine is known to act on GABA receptors).  Genetic LRRC8 mouse models will be essential to elucidate the various postulated and novel cellular and organismal functions of this newly identified channel. The biophysical and physiological analysis of VRAC/VSOAC has become a major focus of our lab.

Own cited publication:

[20] Voss FK, Ullrich F, Münch J, Lazarow K, Lutter D, Mah N, Andrade-Navarro MA, von Kries JP, Stauber T, Jentsch TJ (2014) Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science 344: 634-638

More own publications related to VRAC:

 

Lutter D., Ullrich F., Lueck J.C., Kempa S., Jentsch T.J. (2017). Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels. J. Cell Sci. 130, 1122-1133.

Ullrich F., Reincke S.M., Voss F.K., Stauber T., Jentsch T.J. (2016). Inactivation and anion selectivity of volume-regulated VRAC channels depend on carboxy-terminal residues of the first extracellular loop. J. Biol. Chem., 291, 17040-17048.

Planells-Cases R., Lutter D., Guyader C., Gerhards N.M., Ullrich F., Elger D.A., Kucukosmanoglu A, Xu G., Voss F.K., Reincke S.M., Stauber T., Blomen V.A., Vis D.J., Wessels L.F., Brummelkamp T.R., Borst P., Rottenberg S., Jentsch T.J. (2015). VRAC channel composition determines its substrate specificity and cellular resistance to Pt-based anti-cancer drugs. EMBO J. 34, 2993-3008.

 

Prof. Dr. Thomas Jentsch
Prof. Thomas J. Jentsch
Group Leader
Contact
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) & Max-Delbrück-Centrum für Molekulare Medizin (MDC)
Robert-Rössle-Str. 10
13092 Berlin, Germany
 
Research Lab Coordinator
Dr. Norma Nitschke
+49 30 9406-2974