CAR links hypoxia signaling to improved survival after myocardial infarction.

The coxsackievirus and adenovirus receptor (CAR) mediates homo- and heterotopic interactions between neighboring cardiomyocytes at the intercalated disc. CAR is upregulated in the hypoxic areas surrounding myocardial infarction (MI). To elucidate whether CAR contributes to hypoxia signaling and MI pathology, we used a gain- and loss-of-function approach in transfected HEK293 cells, H9c2 cardiomyocytes and CAR knockout mice. CAR overexpression increased RhoA activity, HIF-1α expression and cell death in response to chemical and physical hypoxia. In vivo, we subjected cardiomyocyte-specific CAR knockout (KO) and wild-type mice (WT) to coronary artery ligation. Survival was drastically improved in KO mice with largely preserved cardiac function as determined by echocardiography. Histological analysis revealed a less fibrotic, more compact lesion. Thirty days after MI, there was no compensatory hypertrophy or reduced cardiac output in hearts from CAR KO mice, in contrast to control mice with increased heart weight and reduced ejection fraction as signs of the underlying pathology. Based on these findings, we suggest CAR as a therapeutic target for the improved future treatment or prevention of myocardial infarction.

Inactivation and Anion Selectivity of Volume-regulated Anion Channels (VRACs) Depend on C-terminal Residues of the First Extracellular Loop.

Canonical volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have many other important roles, including tumor drug resistance and release of neurotransmitters. Although VRAC-mediated swelling-activated chloride currents (ICl,vol) have been studied for decades, exploration of the structure-function relationship of VRAC has become possible only after the recent discovery that VRACs are formed by differently composed heteromers of LRRC8 proteins. Inactivation of ICl,vol at positive potentials, a typical hallmark of VRACs, strongly varies between native cell types. Exploiting the large differences in inactivation between different LRRC8 heteromers, we now used chimeras assembled from isoforms LRRC8C and LRRC8E to uncover a highly conserved extracellular region preceding the second LRRC8 transmembrane domain as a major determinant of ICl,vol inactivation. Point mutations identified two amino acids (Lys-98 and Asp-100 in LRRC8A and equivalent residues in LRRC8C and -E), which upon charge reversal strongly altered the kinetics and voltage dependence of inactivation. Importantly, charge reversal at the first position also reduced the iodide > chloride permeability of ICl,vol This change in selectivity was stronger when both the obligatory LRRC8A subunit and the other co-expressed isoform (LRR8C or -E) carried such mutations. Hence, the C-terminal part of the first extracellular loop not only determines VRAC inactivation but might also participate in forming its outer pore. Inactivation of VRACs may involve a closure of the extracellular mouth of the permeation pathway.

VRAC: molecular identification as LRRC8 heteromers with differential functions.

A major player of vertebrate cell volume regulation is the volume-regulated anion channel (VRAC), which conducts halide ions and organic osmolytes to counteract osmotic imbalances. The molecular entity of this channel was unknown until very recently, although its biophysical characteristics and diverse physiological roles have been extensively studied over the last 30 years. On the road to the molecular identification of VRAC, experimental difficulties led to the proposal of a variety of false candidates. In 2014, in a final breakthrough, two groups independently identified LRRC8A as indispensable component of VRAC. LRRC8A is part of the leucine-rich repeat containing 8 family, which is comprised of five members (LRRC8A-E). Of those, LRRC8A is an obligatory subunit of VRAC but it needs at least one of the other family members to mediate the swelling-induced Cl(-) current ICl,vol. This review discusses the remarkable journey which led to the molecular identification of VRAC, evidence for LRRC8 proteins forming the VRAC pore and their heteromeric assembly. Furthermore, first major insights on the role of LRRC8 proteins in cancer drug resistance and apoptosis and the role of LRRC8D in cisplatin and taurine transport will be summarized.

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs.

Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8-dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug-induced apoptosis independently from drug uptake, possibly by impairing VRAC-dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D-containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.

Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC.

Regulation of cell volume is critical for many cellular and organismal functions, yet the molecular identity of a key player, the volume-regulated anion channel VRAC, has remained unknown. A genome-wide small interfering RNA screen in mammalian cells identified LRRC8A as a VRAC component. LRRC8A formed heteromers with other LRRC8 multispan membrane proteins. Genomic disruption of LRRC8A ablated VRAC currents. Cells with disruption of all five LRRC8 genes required LRRC8A cotransfection with other LRRC8 isoforms to reconstitute VRAC currents. The isoform combination determined VRAC inactivation kinetics. Taurine flux and regulatory volume decrease also depended on LRRC8 proteins. Our work shows that VRAC defines a class of anion channels, suggests that VRAC is identical to the volume-sensitive organic osmolyte/anion channel VSOAC, and explains the heterogeneity of native VRAC currents.