Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes

LRRC8 proteins have been shown to underlie the ubiquitous volume regulated anion channel (VRAC). VRAC channels are composed of the LRRC8A subunit and at least one among the LRRC8B-E subunits. In addition to their role in volume regulation, LRRC8 proteins have been implicated in the uptake of chemotherapeutic agents. We had found that LRRC8 channels can be conveniently expressed in Xenopus oocytes, a system without endogenous VRAC activity. The fusion with fluorescent proteins yielded constitutive activity for A/C, A/D and A/E heteromers. Here we tested the effect of the anticancer drug cisplatin on LRRC8A-VFP/8E-mCherry and LRRC8A-VFP/8D-mCherry co-expressing oocytes. Incubation with cisplatin dramatically activated currents for both subunit combinations, confirming that VRAC channels provide an uptake pathway for cisplatin and that intracellular cisplatin accumulation strongly activates the channels. Thus, specific activators of LRRC8 proteins might be useful tools to counteract chemotherapeutic drug resistance.     

Biophysics and Structure-Function Relationships of LRRC8-Formed Volume-Regulated Anion Channels

Volume-regulated anion channels (VRACs) are key players in regulatory volume decrease of vertebrate cells by mediating the extrusion of chloride and organic osmolytes. They play additional roles in various physiological processes beyond their role in osmotic volume regulation. VRACs are formed by heteromers of LRRC8 proteins; LRRC8A (also called SWELL1) is an essential subunit that combines with any of its paralogs, LRRC8B–E, to form hexameric VRAC complexes. The subunit composition of VRACs determines electrophysiological characteristics of their anion transport such as single-channel conductance, outward rectification, and depolarization-dependent inactivation kinetics. In addition, differently composed VRACs conduct diverse substrates, such as LRRC8D enhancing VRAC permeability to organic substances like taurine or cisplatin. Here, after a recapitulation of the biophysical properties of VRAC-mediated ion and osmolyte transport, we summarize the insights gathered since the molecular identification of VRACs. We describe the recently solved structures of LRRC8 complexes and discuss them in terms of their structure-function relationships. These studies open up many potential avenues for future research.     

LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication

Leucine-rich repeat-containing 8 (LRRC8) proteins are composed of four transmembrane helices and 17 leucine-rich repeats (LRR). Although LRRC8 proteins have been associated with important processes, like maturation of B cells or adipocyte differentiation, their biology and molecular function are largely unknown. We found that LRRC8 proteins originated from the combination of a pannexin and an LRR domain (most likely related to the SHOC2, LAP, RSU1 and LRRIQ4 protein families) before the diversification of chordates. We propose that, like pannexins, LRRC8 proteins form hexameric channels, which participate in cell-cell communication processes. According to the inferred topological model, and contrary to what was previously assumed, the six LRR domains are located in the cytoplasm, and could participate in the organisation of signalling cascades. By compiling available proteomics and gene expression data, and on the basis of the LRRC8 proposed hexameric channel structure, we present clues to the function of this family.     

The Protein Synthesis Inhibitor Blasticidin S Enters Mammalian Cells via Leucine-rich Repeat-containing Protein 8D

Leucine-rich repeat-containing 8 (LRRC8) proteins have been identified as putative receptors involved in lymphocyte development and adipocyte differentiation. They remain poorly characterized, and no specific function has been assigned to them. There is no consensus on how this family of proteins might function because homology searches suggest that members of the LRRC8 family act not as plasma membrane receptors, but rather as channels that mediate cell-cell signaling. Here we provide experimental evidence that supports a role for LRRC8s in the transport of small molecules. We show that LRRC8D is a mammalian protein required for the import of the antibiotic blasticidin S. We characterize localization and topology of LRRC8A and LRRC8D and demonstrate that LRRC8D interacts with LRRC8A, LRRC8B, and LRRC8C. Given the suggested involvement in solute transport, our results support a model in which LRRC8s form one or more complexes that may mediate cell-cell communication by transporting small solutes.     

Deficient of LRRC8A attenuates hypoxia-induced necrosis in 3T3-L1 cells

ABSTRACT

Under acute hypoxia, multiple ion channels on the cell membrane are activated, causing cell swelling and eventually necrosis. LRRC8A is an indispensable protein of the volume-regulated anion channel (VRAC), which participates in swelling and the acceleration of cell necrosis. In this study, we revealed a dynamic change in the expression level of the LRRC8 family during hypoxia in 3T3-L1 cells. The disruption of LRRC8A in 3T3-L1 cells was also associated with a significant anti-necrotic phenotype upon hypoxia accompanied by the reduced expression of necrosis-related genes. In vivo, differential expression of LRRC8 family members was also identified between high-altitude pigs and their low-altitude relatives. Taken these findings together, this study demonstrates the involvement of LRRC8A in hypoxia-induced cell necrosis.     

Distinct contributions of LRRC8A and its paralogs to the VSOR anion channel from those of the ASOR anion channel

Volume- and acid-sensitive outwardly rectifying anion channels (VSOR and ASOR) activated by swelling and acidification exhibit voltage-dependent inactivation and activation time courses, respectively. Recently, LRRC8A and some paralogs were shown to be essentially involved in the activity and inactivation kinetics of VSOR currents in human colonic HCT116 cells. In human cervix HeLa cells, here, inactivation of VSOR currents was found to become accelerated by RNA silencing only of LRRC8A but never decelerated by that of any LRRC8 isoform. These data suggest that LRRC8A is associated with the deceleration mechanism of VSOR inactivation, while none of LRRC8 members is related to the acceleration mechanism. Activation kinetics of ASOR currents was unaffected by knockdown of any LRRC8 family member. Double, triple and quadruple gene-silencing studies indicated that combinatory expression of LRRC8A with LRRC8D and LRRC8C is essential for VSOR activity, whereas none of LRRC8 family members is involved in ASOR activity.     

On the molecular nature of large-pore channels

Membrane transport is a fundamental means to control basic cellular processes such as apoptosis, inflammation, and neurodegeneration and is mediated by a number of transporters, pumps, and channels. Accumulating evidence over the last half century has shown that a type of so-called “large-pore channel” exists in various tissues and organs in gap-junctional and non-gap-junctional forms in order to flow not only ions but also metabolites such as ATP. They are formed by a number of protein families with little or no evolutionary linkages including connexin, innexin, pannexin, leucine-rich repeat-containing 8 (LRRC8), and calcium homeostasis modulator (CALHM). This review summarizes the history and concept of large-pore channels starting from connexin gap junction channels to the more recent developments in innexin, pannexin, LRRC8, and CALHM. We describe structural and functional features of large-pore channels that are crucial for their diverse functions on the basis of available structures.     

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.     

Airway Surface Dehydration by Transforming Growth Factor β (TGF-β) in Cystic Fibrosis Is Due to Decreased Function of a Voltage-dependent Potassium Channel and Can Be Rescued by the Drug Pirfenidone

Transforming growth factor β1 (TGF-β1) is not only elevated in airways of cystic fibrosis (CF) patients, whose airways are characterized by abnormal ion transport and mucociliary clearance, but TGF-β1 is also associated with worse clinical outcomes. Effective mucociliary clearance depends on adequate airway hydration, governed by ion transport. Apically expressed, large-conductance, Ca²⁺- and voltage-dependent K⁺ (BK) channels play an important role in this process. In this study, TGF-β1 decreased airway surface liquid volume, ciliary beat frequency, and BK activity in fully differentiated CF bronchial epithelial cells by reducing mRNA expression of the BK γ subunit leucine-rich repeat-containing protein 26 (LRRC26) and its function. Although LRRC26 knockdown itself reduced BK activity, LRRC26 overexpression partially reversed TGF-β1-induced BK dysfunction. TGF-β1-induced airway surface liquid volume hyper-absorption was reversed by the BK opener mallotoxin and the clinically useful TGF-β signaling inhibitor pirfenidone. The latter increased BK activity via rescue of LRRC26. Therefore, we propose that TGF-β1-induced mucociliary dysfunction in CF airways is associated with BK inactivation related to a LRRC26 decrease and is amenable to treatment with clinically useful TGF-β1 inhibitors.     

Profiling of differentially expressed genes in LRRC4 overexpressed glioblastoma cells by cDNA array

Our previous study has shown that LRRC4 is a novel member of the leucine-rich repeat （LRR） superfamily and has the potential to suppress brain tumor growth. In order to further analyze the functions of LRRC4 on the maintenance of normal function and suppression of tumorigenesis in the central nervous system, we investigated alterations in gene expression related to neurobiology by the Atlas array in two inducible dual-stable LRRC4-overexpressing cell lines. Seventeen of 588 genes spotted on the Atlas membrane showed altered expression levels in LRRC4 transfected U251MG Tet-on cells, which are involved in cell proliferation and cell cycle progression, tumor invasion and metastasis, and neurotransmitter synthesis and release. In addition, cell invasion assay results showed that LRRC4 can inhibit the U251MG cell migration. These studies represent the first cDNA array analysis of the effects of LRRC4 on the involvement of different neurobiological genes in U251MG glioblastoma cells and provide new insights into the function of LRRC4 in glioma.     

VRACs swallow platinum drugs

Platinum‐based drugs such as cisplatin and carboplatin are on the WHO model list of essential medicines, as highly effective chemotherapeutic drugs for the treatment of various solid tumors. These drugs react with purine residues in DNA, thereby causing DNA damage, inhibition of cell division, and eventually cell death. However, the mechanisms whereby platinum‐based drugs enter cancer cells remained poorly understood. In this issue, Planells‐Cases et al ( 2015 ) provide evidence that cells take up cisplatin and carboplatin via volume‐regulated anion channels (VRACs), more specifically VRACs composed of LRRC8A and LRRC8D subunits.     

Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR)

The broadly expressed volume-sensitive outwardly rectifying anion channel (VSOR, also called VRAC) plays essential roles in cell survival and death. Recent findings have suggested that LRRC8A is a core component of VSOR in human cells. In the present study, VSOR currents were found to be largely reduced by siRNA against LRRC8A in mouse C127 cells as well. In contrast, LRRC8A knockdown never affected activities of 4 other types of anion channel activated by acid, Ca²⁺, patch excision or cAMP. While cisplatin-resistant KCP-4 cells poorly expressed endogenous VSOR activity, molecular expression levels of LRRC8A, LRRC8D and LRRC8E were indistinguishable between VSOR-deficient KCP-4 cells and the parental VSOR-rich KB cells. Furthermore, overexpression of LRRC8A alone or together with LRRC8D or LRRC8E in KCP-4 cells failed to restore VSOR activity. These results show that deficiency of VSOR currents in KCP-4 cells is not due to insufficient expression of the LRRC8A/D/E gene, suggesting an essential involvement of some other factor(s), and indicate that further study is required to better understand the complexities of the molecular determinants of VSOR, including the precise role of LRRC8 proteins.     

LRRC8 involved in B cell development belongs to a novel family of leucine-rich repeat proteins

In a previous study, we isolated a novel gene, LRRC8 (leucine-rich repeat-containing 8), in a girl with congenital agammaglobulinemia. We have now identified four unknown LRRC8-like genes, named TA-LRRP, AD158, LRRC5, and FLJ23420. Their predicted structures are very similar to each other, and highly conserved between humans and the mouse. All five genes encode proteins consisting of 16 extracellular leucine-rich repeats (LRRs), all of which have four transmembrane regions except for FLJ23420. These genes belong to a novel family, designated the LRRC8 family, within the superfamily of LRR proteins. TA-LRRP, AD158, and LRRC5 might be implicated in proliferation and activation of lymphocytes and monocytes.     

Expression and functional characterization of LRRC4, a novel brain-specific member of the LRR superfamily

LRRC4, a novel member of LRR superfamily thought to be involved in development and tumorigenesis of the nervous tissue, has the potential to suppress tumorigenesis and cell proliferation of U251MG cells. This study aimed at revealing the correlation between expression of LRRC4 and the maintenance of normal function and tumorigenesis suppression within the central nervous system. We systematically analyzed the expression and tissue distributions of the gene in tissues. Results showed that LRRC4 expression was limited to normal adult brain, both in human and in mouse, and exhibited a development-regulated pattern, but was down-regulated in brain tumor tissues and U251MG cell line. Furthermore, dynamic alterations in gene expression associated with cell cycle progression were investigated by using Tet-on system. Results showed that LRRC4 induced a cell cycle delay at the late G1 phase, probably through the alteration of the expression of different cell cycle regulating proteins responsible for mediating G1-S progression, such as p21(Waf1/Cip1) and p27(Kip1), Cdk2 and PCNA, p-ERK1/2. These findings suggest that LRRC4 may play an important role in maintaining normal function and suppressing tumorigenesis in the central nervous system.     

The leucine-rich repeat domains of BK channel auxiliary γ subunits regulate their expression,trafficking, and channel-modulation functions

As high-conductance calcium- and voltage-dependent potassium channels, BK channels consist of pore-forming, voltage-, and Ca²⁺-sensing α and auxiliary subunits. The leucine-rich repeat (LRR) domain–containing auxiliary γ subunits potently modulate the voltage dependence of BK channel activation. Despite their dominant size in whole protein masses, the function of the LRR domain in BK channel γ subunits is unknown. We here investigated the function of these LRR domains in BK channel modulation by the auxiliary γ1–3 (LRRC26, LRRC52, and LRRC55) subunits. Using cell surface protein immunoprecipitation, we validated the predicted extracellular localization of the LRR domains. We then refined the structural models of mature proteins on the membrane via molecular dynamic simulations. By replacement of the LRR domain with extracellular regions or domains of non-LRR proteins, we found that the LRR domain is nonessential for the maximal channel-gating modulatory effect but is necessary for the all-or-none phenomenon of BK channel modulation by the γ1 subunit. Mutational and enzymatic blockade of N-glycosylation in the γ1–3 subunits resulted in a reduction or loss of BK channel modulation by γ subunits. Finally, by analyzing their expression in whole cells and on the plasma membrane, we found that blockade of N-glycosylation drastically reduced total expression of the γ2 subunit and the cell surface expression of the γ1 and γ3 subunits. We conclude that the LRR domains play key roles in the regulation of the expression, cell surface trafficking, and channel-modulation functions of the BK channel γ subunits.     

LRRC4融合蛋白的构建与表达研究

在前期工作中,采用EST介导的定位候选克隆策略,克隆了一个在脑瘤中表达下调的脑特异表达新基因LRRC4,为进一步研究其结构与功能的关系,构建了含LRRC4基因全长编码区的pGEM-T Easy质粒,在此基础上通过亚克隆构建了LRRC4融合蛋白的绿色荧光蛋白（pEGFP-C1）表达质粒,瞬时转染哺乳动物细胞,结果发现表达的LRRC4融合蛋白定位于活细胞的细胞膜上.同时,构建了LRRC4全长和截短型原核表达pGEX-4T-2质粒,成功而高效地在大肠杆菌BL21 中表达LRRC4融合蛋白.上述工作为制备多抗,深入研究LRRC4基因的功能奠定了基础.     

N-glycosylation of TRPM8 ion channels modulates temperature sensitivity of cold thermoreceptor neurons

TRPM8 is a member of the transient receptor potential ion channel superfamily, which is expressed in sensory neurons and is activated by cold and cooling compounds, such as menthol. Activation of TRPM8 by agonists takes place through shifts in its voltage activation curve, allowing channel opening at physiological membrane potentials. Here, we studied the role of the N-glycosylation occurring at the pore loop of TRPM8 on the function of the channel. Using heterologous expression of recombinant channels in HEK293 cells we found that the unglycosylated TRPM8 mutant (N934Q) displays marked functional differences compared with the wild type channel. These differences include a shift in the threshold of temperature activation and a reduced response to menthol and cold stimuli. Biophysical analysis indicated that these modifications are due to a shift in the voltage dependence of TRPM8 activation toward more positive potentials. By using tunicamycin, a drug that prevents N-glycosylation of proteins, we also evaluated the effect of the N-glycosylation on the responses of trigeminal sensory neurons expressing TRPM8. These experiments showed that the lack of N-glycosylation affects the function of native TRPM8 ion channels in a similar way to heterologously expressed ones, causing an important shift of the temperature threshold of cold-sensitive thermoreceptor neurons. Altogether, these results indicate that post-translational modification of TRPM8 is an important mechanism modulating cold thermoreceptor function, explaining the marked differences in temperature sensitivity observed between recombinant and native TRPM8 ion channels.     

LRRC8A is essential for volume‐regulated anion channel in smooth muscle cells contributing to cerebrovascular remodeling during hypertension

ObjectivesRecent studies revealed LRRC8A to be an essential component of volume‐regulated anion channel (VRAC), which regulates cellular volume homeostasis. However, evidence for the contribution of LRRC8A‐dependent VRAC activity in vascular smooth muscle cells (VSMCs) is still lacking, and the relevant functional role of LRRC8A in VSMCs remains unknown. The primary goal of this study was to elucidate the role of LRRC8A in VRAC activity in VSMCs and the functional role of LRRC8A in cerebrovascular remodeling during hypertension.Materials and MethodssiRNA‐mediated knockdown and adenovirus‐mediated overexpression of LRRC8A were used to elucidate the electrophysiological properties of LRRC8A in basilar smooth muscle cells (BASMCs). A smooth muscle–specific overexpressing transgenic mouse model was used to investigate the functional role of LRRC8A in cerebrovascular remodeling.ResultsLRRC8A is essential for volume‐regulated chloride current (I _Cl, Vol) in BASMCs. Overexpression of LRRC8A induced a voltage‐dependent Cl⁻ current independently of hypotonic stimulation. LRRC8A regulated BASMCs proliferation through activation of WNK1/PI3K‐p85/AKT axis. Smooth muscle‐specific upregulation of LRRC8A aggravated Angiotensin II‐induced cerebrovascular remodeling in mice.ConclusionsLRRC8A is an essential component of VRAC and is required for cell volume homeostasis during osmotic challenge in BASMCs. Smooth muscle specific overexpression of LRRC8A increases BASMCs proliferation and substantially aggravates basilar artery remodeling, revealing a potential therapeutic target for vascular remodeling in hypertension.

The schematic diagram for LRRC8A role in cerebrovascular remodeling. LRRC8A is an essential component of VRAC in BASMCs. During the challenge of hypertension, the activated LRRC8A channel‐mediated‐Cl⁻ efflux increases WNK1 phosphorylation, which in turn triggers AKT phosphorylation and promotes BASMCs proliferation, eventually exacerbates hypertension‐induced cerebrovascular vascular remodeling.     

Aberrant upregulation of LRRC1 contributes to human hepatocellular carcinoma

Loss of apico-basal polarity often results in a malignant phenotype in epithelial tissues. Aberrant expression of polarity mediator proteins is closely associated with this process. LRRC1/LANO, a putative cell polarity regulator, was previously screened from our gene expression profiling in which its expression was significantly upregulated in hepatocellular carcinoma (HCC). In the present study, we provide evidences that LRRC1 plays a potential oncogenic function in HCC. Consistent with the microarray data, quantitative real-time PCR results showed LRRC1 was aberrantly upregulated in 37/56 (66.1 %, more than twofolds) of HCC specimens compared with adjacent non-cancerous livers. Furthermore, the cellular expression of LRRC1 in all HCC cell lines examined exhibited much higher level than that in normal adult liver tissue. Functional analyses revealed that overexpression of LRRC1 promoted, while knockdown of LRRC1 by RNA interference inhibited the growth and colony formation of HCC cells. Importantly, enhanced expression of LRRC1 conferred NIH3T3 cells the ability of cell transformation. In a xenograft tumor model, we found LRRC1 overexpression increased the tumorigenicity of HCC cells. Thus, our collective findings suggest that LRRC1 contributes to HCC development, and may be a potential target for therapeutic intervention in this disease.