4.1 Overall patterns in VRAC research
VRAC-mediated chloride currents have been first described in 1988 (Cahalan and Lewis, 1988; Hazama and Okada, 1988), and subsequent biophysical and cell physiological studies were conducted by several laboratories throughout the 1990s. During this period, various molecular candidates for VRAC were proposed. However, none of these candidates were later proven experimentally to form VRAC (Wine and Luckie, 1996; Strange, 1998; Pu et al., 1999; Stobrawa et al., 2001; Nilius and Droogmans, 2003; Pedersen et al., 2015; Stauber, 2015). A significant breakthrough occurred in 2014 when two independently studies, published almost simultaneously, identified the genes responsible for VRAC through genome-wide RNA silencing screens in high-throughput fluorescence-based assays for VRAC function (Qiu et al., 2014; Voss et al., 2014).
In this study, we employed a combination of bibliometric analyses and network visualizations to provide an overview of the current state of VRAC channel research following its molecular identification in 2014. Our analysis encompassed various aspects, such as the involvement of nations, institutions, authors, and journals in this emerging area. By utilizing these analytical techniques, we aimed to not only describe the existing landscape but also predict the future research interests and hot topics that are likely to continue garnering attention in the VRAC field. This comprehensive approach allows for a deeper understanding of the research trends and dynamics within the VRAC research community, facilitating insights into the direction and potential areas of focus for future investigations.
Typically, the quantity of publications in a specific field serve as a reflection the scientific activities. In the case of VRAC research, we found that 1,501 authors affiliated with 421 institutions in 46 countries or regions have collectively contributed 278 publications from 2014 to 2022. Among these countries, the United States boasted the greatest quantity of publications and citations. Additionally, it maintains the top rank in country-based co-authorship, indicating its significant contribution and collaborative efforts in advancing the field. Over the past 8 years, there has been a significant rise in the number of studies carried out in China, Germany, Japan, and Italy over the past 8 years. Collectively, these countries accounted for 58.6% of all retrieved studies. Germany specifically ranked third in terms of total articles and second in total citations, and ranked fifth in terms of in cooperation with other countries. Furthermore, 421 institutions contributed articles to VRAC research. Among them, the University of Copenhagen from Denmark as the most prolific institution, with 16 published papers related to VRAC.
4.2 Influential authors and publications in VRAC research
Thomas J. Jentsch of the Max Delbrück Center for Molecular Medicine and the Leibniz-Forschungsinstitut für Molekulare Pharmakologie in Berlin, Germany, holds the records for the highest number of publications and citations. His research team is concerned with ion transport in the broadest sense. Their current endeavors revolve around unraveling the intricate relationship between structure and function in VRAC-LRRC8 proteins, aiming to elucidate their roles in different biological processes and diseases. After the molecular identification of VRAC (Voss et al., 2014), his group has demonstrated that VRAC participates in a wide array of processes, including resistance to cancer drugs (Planells-Cases et al., 2015), secretion of insulin by pancreatic β-cells (Stuhlmann et al., 2018), enhancing innate immunity by transporting the messenger molecule cGAMP (Zhou C. et al., 2020) and proximal tubules function and integrity (López-Cayuqueo et al., 2022). It has also contributed with studies on the structure-function relationship of VRAC (Ullrich et al., 2016; Zhou et al., 2018). Simultaneously, his team explores several other directions, including CLC chloride channels (Jentsch and Pusch, 2018), and the recently identified acid-activated anion channel ASOR (Yang et al., 2019a; Ullrich et al., 2019).
In terms of citation analysis and co-cited of references, it has been revealed that the seminal works published by Voss et al. in Science (Voss et al., 2014) and Qiu et al. in Cell (Qiu et al., 2014) accumulated the highest number of citations, not only because they are the earliest studies in the investigated time frame. These two publications separately discovered the genes responsible for encoding VRAC. LRRC8A, an essential VRAC subunit, was identified in both studies (Qiu et al., 2014; Voss et al., 2014). Voss et al. additionally revealed that VRACs are heteromeric channels consisted of LRRC8A and at least one other member from the LRRC8 family (LRRC8B-E) (Voss et al., 2014), subsequently also confirmed by the Patapoutian lab (Syeda et al., 2016). Surprisingly, the excessive expression of functional LRRC8 heteromers does not lead to an elevation of VRAC currents beyond the levels observed in the endogenous state. In fact, the over-expression of LRRC8A alone has the effect of reducing VRAC activity (Qiu et al., 2014; Voss et al., 2014).
4.3 Research hotspots and Frontiers
Our analysis of co-occurrence networks, which were organized based on topic area or publication date, have provided insight into the current hotspots and potential future directions in VRAC research (Figure 8). According to our keyword co-occurrence analysis, VRAC is implicated in various physiological processes, including but not limited to cell growth, movement, programmed cell death, swelling, and myogenesis. The transport of anions and organic osmolytes such as glutamate, taurine and ATP is part of its function. VRAC has also exhibited connections with other ion channels, including TMEM16A, TMEM16F, pannexin, and CFTR. It has been associated with various diseases including epilepsy, leukodystrophy, atherosclerosis, hypertension, diabetes, cerebral edema and stroke. It has been connected to different types of cancer including gastric cancer, glioblastoma, hepatocellular carcinoma and it has been implicated in anti-tumor drug resistance by its role in cellular drug uptake. In addition to general questions regarding the biophysics and biochemistry of VRAC, research on VRAC will be on particular physiological processes, each of which will address specific questions about the respective role of VRAC. Additionally, the pharmacology of VRAC has been studied. Here, we just highlight some examples.
4.3.1 Activation mechanism of VRAC
Despite extensive investigation, the mechanism responsible for activating VRAC is still obscure and has remained one of the main questions to be addressed on a structure-functional, biochemical, cell biological and physiological level. A number of studies proposed that VRAC gating could be influenced or triggered by different stimuli, molecules within the cell, second messengers, and pathways of signaling. It is worth noting that a significant amount of research on VRAC activation was carried out before the identification of its molecular components (Stauber, 2015; Pedersen et al., 2016; Strange et al., 2019; Bertelli et al., 2021). The identification of LRRC8 heteromers as essential constituents allows using an increasing repertoire of molecular biological tools (Qiu et al., 2014; Voss et al., 2014; Kolobkova et al., 2021). In particular, the determination of LRRC8 complexes structures (Deneka et al., 2018; Kasuya et al., 2018; Kefauver et al., 2018; Kern et al., 2019; Nakamura et al., 2020; Deneka et al., 2021; Kern et al., 2023; Rutz et al., 2023; Takahashi et al., 2023) and other studies on the subunit composition and structure-function relationships (Gaitán-Peñas et al., 2016; Ullrich et al., 2016; Lutter et al., 2017; Schober et al., 2017; Yamada and Strange, 2018; Zhou et al., 2018; Pervaiz et al., 2019; Yamada et al., 2021; Bertelli et al., 2022), will greatly contribute to the understanding of the VRAC activation mechanism(s).
Intracellular ionic strength was implicated in regulating VRAC activity already before the molecular identification of this channel (Strange et al., 2019). Syeda et al. demonstrated that reduced intracellular ionic strength activated reconstituted VRAC in lipid droplet bilayer (Syeda et al., 2016) and reduced ionic strength has subsequently been utilized to activate VRAC currents in various electrophysiological studies. Several observations hint towards a role of the leucin-rich repeat domains (LRRDs) in VRAC gating: increased basal channel activity when proteins are fused to the C-terminal LRRDs (Gaitán-Peñas et al., 2016), a rearrangement of these domains during channel activation as shown by FRET measurements (König et al., 2019) and activating or inhibiting effect of synthetic nanobodies binding to the LRRDs (Deneka et al., 2021). As the LRRDs contain a large amount of charged amino acids, it has been hypothesized that reduced ionic strength changes the electrostatic interaction between the LRRDs within channel complex, thereby leading to VRAC opening. However, the reduction of ionic strength required to activate VRAC is much greater than that expected upon hypotonic activation; therefore, it is improbable that low intracellular ionic intensity serves as the physiologically significant trigger (Strange et al., 2019). Moreover, VRAC can be activated regardless of alterations in ionic concentration or extracellular osmolarity, such as through the use of sphingosine-1-phosphate or cisplatin (Burow et al., 2015; Planells-Cases et al., 2015; Gradogna et al., 2017a). Consistently, using FRET sensor within an intra-complex for monitoring VRAC activity, König et al. found that VRAC activity was not strictly coupled to reduced intracellular ionic strength (König et al., 2019).
Phosphorylation events were also implicated in the cellular signal transduction to VRAC gating (Bertelli et al., 2021). A number of studies have examined the impact of the protein kinase C (PKC) family on VRAC activity, yielding partly contradicting results as to its modulating effects on VRAC activity. For example, some studies have demonstrated that conventional PKC isoforms, specifically PKCα and PKCβI contributed to VRAC activation which induced by ATP (Rudkouskaya et al., 2008). PKCα has been demonstrated to have a function in controlling cell volume during hypotonic swelling (Senju et al., 2015). However, in human glioblastoma (GBM) cells, PKC activity was found to have no effect on VRAC gating (Catacuzzeno et al., 2014).
Another event that has been proposed as an underlying factor in VRAC gating is oxidation (Bertelli et al., 2021; Friard et al., 2021). Gradogna et al. demonstrated that VRAC is directly regulated by oxidation, and this regulation is dependent on the composition LRRC8 subunits (Gradogna et al., 2017b). Specifically, oxidation suppressed the activity of LRRC8A/LRRC8C and LRRC8A/LRRC8D, while it strongly potentiated the activity of LRRC8A/LRRC8E heteromers through intracellular cysteines oxidation (Gradogna et al., 2017a). Bertelli and others have recently discovered that oxidation targets two cysteines, namely, C424 and C448, which are situated in the initial two leucine rich repeats of LRRC8E. In addition, it was found that the oxidation of LRRC8C’s initial methionine hinders the functioning of VRAC (Bertelli et al., 2022). Conversely, VRAC was in turn shown to affect the cellular reactive oxygen species (ROS) levels, possibly through the release of reduced glutathione (Friard et al., 2019).
4.3.2 The (patho)-physiological role of VRAC in the brain
VRAC is an activated channel that responds to cellular swelling and involved in regulateing cell volume reduction in most vertebrate cells, including those found in the central nervous system (CNS). In the brain, the role of VRAC extends to both normal and abnormal states (Akita and Okada, 2014; Mongin, 2016; Elorza-Vidal et al., 2019). For instance, VRAC has been implicated in the modulation of cell volume changes in astrocytes observed in megalencephalic leukoencephalopathy with cysts (MLC), which is associated with mutations in MLC1 or GlialCAM. However, the expression and subcellular distribution of LRRC8A, remain unaffected in mice with disrupted MLC1 or GlialCAM (Bugiani et al., 2017; Elorza-Vidal et al., 2018).
Mogin et al., proposed that VRAC in astrocytes and microglia may have significant physiological and detrimental effect by releasing excitatory amino acids (EAAs) like glutamate and aspartate in both normal and pathological conditions (Mongin, 2016). The release of astrocytic EAAs is considered to promote physiological interactions between astrocytes and neurons (Akita and Okada, 2014; Mongin, 2016; Elorza-Vidal et al., 2019). However, during brain ischemia, the release of EAAs by astrocytes is believed to contributed to neuronal cell death via excitotoxicity (Mongin, 2016). VRAC was reported to regulate EAA release in cultured astrocytes under swelling-induced and isovolumetric conditions (Hyzinski-García et al., 2014; Schober et al., 2017). Different subunits of VRAC are responsible for the release of specific EAAs, with LRRC8D facilitating the efflux of uncharged osmolytes and the LRRC8C/E combination was found to be crucial for the conductance of glutamate and aspartate (Lutter et al., 2017; Schober et al., 2017).
Research utilizing LRRC8A conditional knockout mice have provided insights regarding the role of VRAC in astrocytes during brain injury. These mice showed significantly reduced the volume of brain infarct and improved the score of neurological severity in a stroke model known as temporal middle cerebral artery occlusion (tMCAO), indicating that VRAC channel in astrocytes is indeed participated in brain damage which induced by stroke in vivo (Yang et al., 2019b; Zhou J. J. et al., 2020). Moreover, Zhou et al. reported that during cerebral ischemia, there is an elevation in neuronal VRAC currents that dependent on LRRC8A. This enhanced VRAC activity promotes the influx of glutamatergic to hippocampal neurons, contributing to ischemic stroke-induced brain injury (Zhou J. J. et al., 2020). In addition, the Qiu group recently found that VRAC-mediated ATP release from microglia in spinal cord is a key determinant of neuropathic pain (Chu et al., 2023).
4.3.3 VRAC may represent a novel cancer therapeutic target
Ion channel dysfunction is increasingly associated with cancer (Prevarskaya et al., 2018). Among them, VRAC has been suggested to play a role in regulating cell growth, movement and apoptosis of tumor cells (Fraser and Pardo, 2008; Arcangeli and Becchetti, 2015; Sirianant et al., 2016; Prevarskaya et al., 2018). Downregulation of LRRC8A subunit has been reported to suppress the growth or proliferation of different cancer cell types, including human glioblastoma cells (Rubino et al., 2018), colorectal carcinoma cells (Fujii et al., 2018), esophageal squamous cell carcinoma cells (Konishi et al., 2019), gastric cancer (Kurashima et al., 2021), colon cancer (Zhang et al., 2018; Zhang et al., 2023) and cervical cancer (Chen et al., 2023), while LRRC8A overexpression reportedly facilitated hepatocellular carcinoma cell growth (Lu et al., 2019). In vivo studies in nude mouse models have also shown that LRRC8A downregulation suppressed the proliferation of colon carcinoma, hepatocellular carcinoma, and cervical cancer (Zhang et al., 2018; Lu et al., 2019; Chen et al., 2023).
Cell migration occurs during the whole cascade of cancer development and is particularly critical during metastasis. Local changes in cell volume, driven by a variety of ion transporters or channels, have been linked with and may even propel cell migration (Stroka et al., 2014). VRAC has been suggested to be one of these ion channels. Indeed, while pharmacological or molecular biological inhibition of VRAC did not detectably impair migration of various cell types in a study using a wound-healing assay (Liu and Stauber, 2019), other studies reported VRAC influenced the motility of a wide range of cancer cells such as colorectal cancer (Fujii et al., 2018), glioblastoma (Rubino et al., 2018; Wong et al., 2018), human colon cancer (Zhang et al., 2018), hepatocellular carcinoma (Lu et al., 2019), pancreatic adenocarcinoma (Xu et al., 2022) and breast cancer (Zhang et al., 2022).
Cancer cells frequently acquire multidrug resistance. VRAC was shown to be mediate cellular internalization of platinum-containing chemotherapeutic agents such as cisplatin and carboplatin into cells. The ability of these drugs to pass through VRAC is dependent on the presence of LRRC8D. Interestingly, when either LRRC8A or LRRC8D is knocked out, cancer cells exhibit a higher tolerance towards these platinum-based medications (Planells-Cases et al., 2015). LRRC8 genes have been implicated in drug resistance and prognosis in various cancer types (Sørensen et al., 2016; Xu et al., 2020; Siemer et al., 2021; Widmer et al., 2022; Xu et al., 2022).
4.3.4 Pharmacology of VRAC
Given the involvement of VRAC in various physiological and pathological processes, of which we have covered only a few, the investigation of compounds that modify its activity is an important research field. For example, besides cancer and ischemic conditions after stroke, VRAC modulators were recently reported to be beneficial for murine type 2 diabetes (Gunasekar et al., 2022).
DCPIB (4-(2-butyl-6,7-dichlor-2-cyclopentyl- indan-1-on-5-yl)oxybutyric acid) has been identified as a highly potent selective VRAC inhibitor (Decher et al., 2001). Numerous studies have demonstrated that DCPIB effectively restrain VRAC activity in various types of cells, including HCT116, HeLa, HEK293, Xenopus oocytes, calf bovine pulmonary artery endothelial cells, Guinea-pig atrial cardiomyocytes, rat pancreatic beta cells and mouse astrocytes (Decher et al., 2001; Harrigan et al., 2008; Bantel et al., 2009; Akita and Okada, 2011; Schlichter et al., 2011; Stott et al., 2014; Yamada et al., 2021; Figueroa and Denton, 2022). DCPIB distinguishes itself from other VRAC blockers owing to its exceptional specificity for the VRAC channel, selectively targeting it over other chloride channels such as Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), Calcium-activated Chloride Channels (CaCCs), and Proton-Activated Chloride (PAC), CLCs and Maxi-Cl channels (Decher et al., 2001; Sabirov and Okada, 2005; Sabirov et al., 2016; Sato-Numata et al., 2016). However, it experienced off-target effects on various transporters and channels including glutamate transporter GLT-1, Kir and K2P potassium channels, connexin hemichannels, H+,K+-ATPase (Bowens et al., 2013; Fujii et al., 2015; Deng et al., 2016; Lv et al., 2019). Moreover, DCPIB has been found to trigger K2P K+ channels, particularly TREK1, TREK2 and TRAAK (Minieri et al., 2013; Lv et al., 2019). Additionally, it has been demonstrated to induced the activation of BK K+ channels (Zuccolini et al., 2022).
To identify further modulators of VRACs, Figueroa et al. recently screened 1,184 FDA-approved drugs and found that the cysteinyl leukotriene receptor 1 (CysLT1R) antagonists pranlukast and zafirlukast blocked VRAC currents in HEK293 cells in a dose-dependent manner (Figueroa et al., 2019; Figueroa and Denton, 2022). Importantly, VRAC inhibition by pranlukast and zafirlukast was independent of their known target CysLT1R. Furthermore, Xue et al. demonstrated that both natural and synthetic flavonoids can inhibit endogenous VRAC currents in HEK293 and HUVEC cells (Xue et al., 2018). Although these compounds are not fully specific to VRAC, their discovery as blockers of VRAC activity provide a valuable chemical framework for the creation of improved and targeted VRAC inhibitors.
Zinc pyrithione (ZPT), commonly utilized as an antifouling agent and for addressing conditions like dandruff and other skin disorders, was described as an activator of VRAC (Figueroa and Denton, 2021). In addition to enhances the rate of VRAC currents caused by cell swelling, ZPT also potentiated those currents even without cell swelling (Figueroa and Denton, 2021). In the future, it is necessary to study the mechanism of ZPT in activating VRAC, which will provide valuable insights into comprehending the relationship between channel structure, function and regulatory mechanisms.