Novel identification and characterisation of Transient receptor potential melastatin 3 ion channels on Natural Killer cells and B lymphocytes: effects on cell signalling in Chronic fatigue syndrome/Myalgic encephalomyelitis patients.

BACKGROUND: Transient receptor potential melastatin 3 (TRPM3) cation channels are ubiquitously expressed by multiple cells and have an important regulatory role in calcium-dependent cell signalling to help maintain cellular homeostasis. TRPM3 protein expression has yet to be determined on Natural Killer (NK) cells and B lymphocytes. Multiple single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) patients and have been proposed to correlate with illness presentation. The object of the study was to assess TRPM3 surface expression on NK and B lymphocytes from healthy controls, followed by a comparative investigation examining TRPM3 surface expression, and cytoplasmic and mitochondrial calcium influx in CD19(+) B cells, CD56(bright) and CD56(dim) cell populations from CFS/ME patients. RESULTS: TRPM3 cell surface expression was identified for NK and B lymphocytes in healthy controls (CD56(bright) TRPM3 35.72 % ± 7.37; CD56(dim) 5.74 % ± 2.00; B lymphocytes 2.05 % ± 0.19, respectively). There was a significant reduction of TRPM3 surface expression on CD19(+) B cells (1.56 ± 0.191) and CD56(bright) NK cells (17.37 % ± 5.34) in CFS/ME compared with healthy controls. Anti-CD21 and anti-IgM conjugated biotin was cross-linked with streptavidin,and subsequently treatment with thapsigargin. This showed a significant reduction in cytoplasmic calcium ion concentration in CD19(+) B lymphocytes. CD56(bright) NK cells also had a significant decrease in cytoplasmic calcium in the presence of 2-APB and thapsigargin in CFS/ME patients. CONCLUSIONS: The results from this preliminary investigation identify, for the first time, TRPM3 surface expression on both NK and B lymphocytes in healthy controls. We also report for the first time, significant reduction in TRPM3 cell surface expression in NK and B lymphocytes, as well as decreased intracellular calcium within specific conditions in CFS/ME patients. This warrants further examination of these pathways to elucidate whether TRPM3 and impaired calcium mobilisation has a role in CFS/ME.

Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

Role of caveolar compartmentation in endothelium-derived hyperpolarizing factor-mediated relaxation: Ca2+ signals and gap junction function are regulated by caveolin in endothelial cells.

BACKGROUND: In endothelial cells, caveolin-1, the structural protein of caveolae, acts as a scaffolding protein to cluster lipids and signaling molecules within caveolae and, in some instances, regulates the activity of proteins targeted to caveolae. Specifically, different putative mediators of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation are located in caveolae and/or regulated by the structural protein caveolin-1, such as potassium channels, calcium regulatory proteins, and connexin 43, a molecular component of gap junctions. METHODS AND RESULTS: Comparing relaxation in vessels from caveolin-1 knockout mice and their wild-type littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from caveolin-1 knockout mice. The absence of caveolin-1 is associated with an impairment of calcium homeostasis in endothelial cells, notably, a decreased activity of Ca2+-permeable TRPV4 cation channels that participate in nitric oxide- and EDHF-mediated relaxation. Moreover, morphological characterization of caveolin-1 knockout and wild-type arteries showed fewer gap junctions in vessels from knockout animals associated with a lower expression of connexins 37, 40, and 43 and altered myoendothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolar compartment of the plasma membrane. CONCLUSIONS: We demonstrated that expression of caveolin-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway.

On the putative role of transient receptor potential cation channels in asthma.

The mammalian transient receptor potential (TRP) superfamily consists of 28 mammalian TRP cation channels, which can be subdivided into six main subfamilies: the TRPC ('Canonical'), TRPV ('Vanilloid'), TRPM ('Melastatin'), TRPP ('Polycystin'), TRPML ('Mucolipin') and the TRPA ('Ankyrin') groups. Increasing evidence has accumulated during the previous few years that links TRP channels to the cause of several diseases or to critically influence and/or determine their progress. This review focuses on the possible role of TRP channels in the aetiology of asthmatic lung disease.

Structure-function relationship of the TRP channel superfamily.

Transient receptor potential (TRP) channels are involved in the perception of a wide range of physical and chemical stimuli, including temperature and osmolarity changes, light, pain, touch, taste and pheromones, and in the initiation of cellular responses thereupon. Since the last decade, rapid progress has been made in the identification and characterization of new members of the TRP superfamily. They constitute a large superfamily of cation channels that are expressed in almost all cell types in both invertebrates and vertebrates. This review summarizes and discusses the current knowledge on the TRP protein structure and its impact on the regulation of the channel function.

Effect of prolonged c-kit receptor inhibition by imatinib mesylate on the uterine contractility of pregnant rabbits.

BACKGROUND: The c-kit receptor expressed by interstitial cells in the gastrointestinal tract is crucial to their pacemaking function. The function of similar c-kit-expressing myometrial cells is unknown. METHODS: Imatinib mesylate, a specific c-kit receptor antagonist, was administered to pregnant New Zealand white rabbits (term = 31 days, n = 35) from day 27 gestation by intramuscular injection twice daily at high (50 microg/kg) or medium (10 microg/kg) dose and compared with a control group injected with vehicle only. In a second phase, two further groups received imatinib at medium or low (1 mug/kg) dose for a longer duration starting from day 18 until delivery. Three does from the latter groups as well as controls underwent myometrial biopsy under general anesthesia after spontaneous vaginal birth. Contractility was recorded by isometric tensiometry. The outcome measures were delay of parturition and in vitro contractility characteristics. RESULTS: High-dose imatinib induced early delivery when compared with the control group (28.6 vs. 30.7 days, p < 0.001). The other groups delivered at term. No effect on in vitro contractility was apparent in any of the groups. CONCLUSIONS: c-kit receptor inhibition in pregnant rabbits does not delay significantly the length of gestation or change myometrial contractility in vitro.

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration.

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.

Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation.

In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn locus in the CFTR (cystic fibrosis transmembrane conductance regulator) gene is a frequent disease mutation with incomplete penetrance. This T5 allele will result in a high proportion of CFTR transcripts that lack exon 9, whose translation products will not contribute to apical chloride channel activity. Besides the polymorphic Tn locus, more than 120 polymorphisms have been described in the CFTR gene. We hypothesized that the combination of particular alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. Analysis of three polymorphic loci with frequent alleles in the general population showed that, in addition to the known effect of the Tn locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci: (TG)m and M470V. On a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, and (TG)12 gave a sixfold increase, compared with the (TG)10 allele. T5 CFTR genes derived from patients were found to carry a high number of TG repeats, while T5 CFTR genes derived from healthy CF fathers harbored a low number of TG repeats. Moreover, it was found that M470 CFTR proteins matured more slowly, and that they had a 1.7-fold increased intrinsic chloride channel activity compared with V470 CFTR proteins, suggesting that the M470V locus might also play a role in the partial penetrance of T5 as a disease mutation. Such polyvariant mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations, and be of relevance in other genetic diseases.

Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium.

TRPV4 is a broadly expressed Ca2+-permeable cation channel in the vanilloid subfamily of transient receptor potential channels. TRPV4 gates in response to a large variety of stimuli, including cell swelling, warm temperatures, the synthetic phorbol ester 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), and the endogenous lipid arachidonic acid (AA). Activation by cell swelling and AA requires cytochrome P450 (CYP) epoxygenase activity to convert AA to epoxyeicosatrienoic acids (EETs) such as 5,6-EET, 8,9-EET, which both act as direct TRPV4 agonists. To evaluate the role of TRPV4 and its modulation by the CYP pathway in vascular endothelial cells, we performed Ca2+ imaging and patch-clamp measurements on mouse aortic endothelial cells (MAECs) isolated from wild-type and TRPV4(-/-) mice. All TRPV4-activating stimuli induced robust Ca2+ responses in wild-type MAECs but not in MAECs isolated from TRPV4(-/-) mice. Upregulation of CYP2C expression by preincubation with nifedipine enhanced the responses to AA and cell swelling in wild-type MAECs, whereas responses to other stimuli remained unaffected. Conversely, inhibition of CYP2C9 activity with sulfaphenazole abolished the responses to AA and hypotonic solution (HTS). Moreover, suppression of EET hydrolysis using 1-adamantyl-3-cyclo-hexylurea or indomethacin, inhibitors of soluble epoxide hydrolases (sEHs), and cyclooxygenases, respectively, enhanced the TRPV4-dependent responses to AA, HTS, and EETs but not those to 4alpha-PDD or heat. Together, our data establish that CYP-derived EETs modulate the activity of TRPV4 channels in endothelial cells and shows the unraveling of novel modulatory pathways via CYP2C modulation and sEH inhibition.

Insights into TRPM4 function, regulation and physiological role.

In the current review we will summarise data from the recent literature describing molecular and functional properties of TRPM4. Together with TRPM5, these channels are up till now the only molecular candidates for a class of non-selective, Ca(2+)-impermeable cation channels which are activated by elevated Ca2+ levels in the cytosol. Apart from intracellular Ca2+, TRPM4 activation is also dependent on membrane potential. Additionally, channel activity is modulated by ATP, phosphatidylinositol bisphosphate (PiP2), protein kinase C (PKC) phosphorylation and heat. The molecular determinants for channel activation, permeation and modulation are increasingly being clarified, and will be discussed here in detail. The physiological role of Ca(2+)-activated non-selective cation channels is unclear, especially in the absence of gene-specific knock-out mice, but evidence indicates a role as a regulator of membrane potential, and thus the driving force for Ca2+ entry from the extracellular medium.

TRPM8.

Originally cloned as a prostate-specific protein, TRPM8 is now best known as a cold- and menthol-activated channel implicated in thermosensation. In this chapter we provide a brief review of current knowledge concerning the biophysical properties, gating mechanisms, pharmacology and (patho)physiology of this TRP channel.

Modulation of cardiac Na channels by angiotensin II.

The modulation of Na channels by the vasoactive peptide angiotensin II (AT II) has been studied in isolated ventricular cells of guinea pigs using the patch clamp technique. In cell-attached patches the maximal probability of the channel being open was increased in a concentration range between 0.05 and 1 microM, but decreased at higher concentrations. A maximal increased of 2.5 +/- 0.86 was found at 1 microM AT II. The increase in the probability of the channel being open was due to a decrease in the number of nulls. In all affected cells (n = 17) we observed a delayed inactivation after application of AT II at concentrations between 0.05 and 10 microM. At -30 mV, the time constant of inactivation increased from 1.1 +/- 0.1 ms (controls) to 5.6 +/- 1.6 ms (10 microM AT II). This effect was due to an increased number of openings per sweeps. No significant effect on the mean open time and the first latency were observed. However, due to pronounced bursting, the averaged closed time was significantly increased from 0.8 +/- 0.1 ms to 1.3 +/- 0.1 ms in the presence of 1 microM AT II at -30 mV. An effect of AT II on cardiac Na channels via protein kinase C is discussed.

The conductance of single cardiac sodium channels from guinea pig depends on the intracellular sodium concentration.

Currents through DPI 201-106 modified single sodium channels have been measured in cell-free inside-out patches from guinea-pig ventricular myocytes. Single-channel conductance and reversal potential of the sodium channel have been calculated at different intracellular sodium concentrations [( Na+]i) from microscopic I-V curves, which were obtained by application of linear voltage ramps. The relation between the reversal potential and [Na+]i could be fitted with a modified Goldman-Hodgkin-Katz equation with a relative permeability for K+ over Na+ ions of 0.054. The zero-current conductance of the Na channel as a function of [Na+]i shows a plateau value at low Na concentrations, and increases in a sigmoidal manner at higher concentrations. It is concluded that the Na channel can carry outward currents and that its conductance depends on [Na+]i.

Amplitude modulation of Ca2+ signals induced by histamine in human endothelial cells.

We have addressed the problem of whether the agonist concentration sensed by endothelial cells is encoded by the sustained rise of the intracellular Ca2+ concentration ([Ca2+]i) or by the frequency of intracellular Ca2+ oscillations. Single or confluent endothelial cells from umbilical veins were stimulated for 15 min with histamine (0.03 to 100 mumol/l), and the concomitant changes in [Ca2+]i were measured with fura-2/AM. Application of histamine at concentrations above 0.1 mumol/l resulted always in a fast spike of [Ca2+]i, followed by a slow decline to a sustained plateau level, which depends on the presence of extracellular Ca2+. At the same time of the development of this plateau phase, quenching of the fura-2/AM signal occurred during agonist stimulation in a Ca(2+)-free, 1.5 mmol/l Mn2+ containing solution, indicating influx of divalent cations during this time. From 48 cells in 1.5 mmol/l [Ca2+]e we obtained a close relation between histamine concentration and time integral of [Ca2+]i taken over the 15 min recording of the plateau [Ca2+]i. The half-maximal increase in the integral of [Ca2+]i was at 0.7 mumol/l for solitary cells, 1.2 mumol/l for clustered cells and 1.2 mumol/l for the plateau Ca2+ level. Repetitive Ca2+ spikes or Ca2+ oscillations appeared only in 16 out of 48 cells, but their frequency was not correlated to the agonist concentration. Ca2+ oscillations were only observed in a concentration window between 0.1 and 1 mumol/l histamine, both in single and in clustered endothelial cells. Our results indicate that coding of the agonist concentration in endothelial cells is not related to the frequency of Ca2+ oscillations, but is closely correlated with the plateau level of intracellular Ca2+.

Mechanical stress induces release of ATP from Ehrlich ascites tumor cells.

The supernatant from a suspension of Ehrlich cells exposed to centrifugation at 700xg for 45 s induced a transient increase in the intracellular concentration of free, cytosolic Ca2+, [Ca2+]i, as well as activation of an outwardly rectifying whole-cell current when added to a suspension of non-stimulated cells. These effects were inhibited by suramin, a non-specific P2 receptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revealed that the supernatant from Ehrlich cells exposed to centrifugation contained 2. 6+/-0.2 microM ATP, and that the mechanical stress-induced release of ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprotein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a suramin-sensitive, transient, concentration-dependent increase in [Ca2+]i, activation of an outwardly rectifying whole-cell current and a hyperpolarization of the plasma membrane. The ATP-induced hyperpolarization of the plasma membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca2+-activated K+ channels, suggesting that stimulation of P2 receptors in Ehrlich cells evokes a Ca2+-activated K+ current. The relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, alpha,beta-MeATP, bzATP) in eliciting an increase in [Ca2+]i, as well as the effect of repetitive addition of nucleotides were investigated. The results lead us to conclude that mechanical stimulation of Ehrlich cells leads to release of ATP, which in turn stimulates both P2Y1 and P2Y2 receptors, resulting in Ca2+ influx as well as release and activation of an outwardly rectifying whole-cell current.

Modulation of the T-type cardiac Ca channel by changes in proton concentration.

Single-channel measurements and whole-cell experiments with the two suction electrode, voltage clamp technique were used to investigate the effects of external and internal proton concentrations on T-type Ca channels in heart muscle cells of the guinea pig. As in the L-type Ca channel, an increase in the external proton concentration decreases T-type currents, while external alkalinization enlarges the currents. In contrast to the L-type Ca channel, however, a change in the internal proton concentration does not modulate T-type Ca currents. The T-type Ca channel is much more sensitive to variations in pHo than the L-type Ca channel. By the combination of single-channel and whole-cell experiments we can conclude that the observed changes in macroscopic currents are due to (a) changes in the single-channel conductance and in the probability of the T-type Ca channel being open, and (b) the titration of the negative surface charges in the neighborhood of the T-type Ca channel with shifts of both the activation and inactivation processes of the channel. The pHo-induced changes in the maximal conductance (gmax) of the T-type Ca channel show an apparent pKa in the range of 7.1-7.5, while the titration of the negative surface charges near the channel shows an apparent pKa of 7.1 with a concomitant surface potential of -24.6 mV at 5.4 mM [Ca]o. These pKa values, less acid than the pKa values found for the pHo-induced, L-type Ca channel modulation, might imply a physiological importance of this novel type of channel modulation.

Modulation of voltage-dependent properties of a swelling-activated Cl- current.

We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl- current (ICl,swell) in BC3H1 myoblasts. This Cl- current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl- currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (approximately 60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10-80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg2+ concentration or reducing the Cl- concentration of the extracellular solution. Replacing extracellular Cl- by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN- > I- > NO3- > Cl- >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg2+, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of ICl,swell are strongly influenced by external pH, external divalent cations, and by the nature of the permeant anion.

Activation of a Cl- current by hypotonic volume increase in human endothelial cells.

We have used whole-cell and perforated patches to study ionic currents induced by hypotonic extracellular solutions (HTS, 185 mOsm instead of 290 mOsm) in endothelial cells from human umbilical veins. These currents activated within 30-50 s after application of HTS, reached a maximum value after approximately 50-150 s and recovered completely after re-exposing the cells to normal osmolarity. They slowly inactivated at potentials positive to +50 mV. The same current was also activated by breaking into endothelial cells with a hypertonic pipette solution (377 mOsm instead of 290 mOsm). The reversal potential of these volume-induced currents using different extracellular and intracellular Cl- concentrations was always close to the Cl(-)-equilibrium potential. These currents are therefore mainly carried by Cl-. DIDS only weakly blocked the current (KI = 120 microM), while another Cl(-)-channel blocker, DCDPC (20 microM) was ineffective. We were unable to record single channel activity in cell-attached patches but we always observed an increased current variance during HTS. From the mean current-variance relation of the whole-cell current records, we determined a single channel conductance of 1.1 pS. The size and kinetics of the current were not correlated with the concomitant changes in intracellular calcium. Furthermore, the currents could still be activated in the presence of 10 mmol/liter intracellular EGTA and are thus Ca2+ independent. A similar current was also activated with iso-osmotic pipette solutions containing 300 mumol/liter GTP gamma S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 mumol/liter) was also ineffective in modulation of this current. The HTS-induced current was completely blocked by 10 mumol/liter pBPB, a PLA2 inhibitor. NDGA (4 mumol/liter) and indomethacin (5 mumol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 mumol/liter) were variable. In 12 out of 40 cells it either directly activated a Cl- current or potentiated the current activated by HTS. The membrane current was decreased at all potentials in 18 cells, and was not affected in 10 cells. The HTS-induced currents may therefore be modulated by cleavage products of PLA2, but not by messengers downstream of arachidonic acid. Loading the cells with a segment of the heat stable protein kinase A inhibitor PKI (5-24) did not prevent activation of the HTS-induced current.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular mechanisms of L-selectin induced capping.

Leucocyte adhesion to endothelial cells is a tightly regulated process involving selectins, integrins and immunoglobulin-like proteins. Cell adhesion and communication are controlled by membrane dynamics like receptor capping. Capping of surface receptors is an ubiquitous mechanism but still not well understood. Employing immunofluorescence techniques, we demonstrate that L-selectin triggering results in receptor capping of the L-selectin molecules in lymphocytes. Using pharmacological inhibitors and genetic deficient cell lines we show that this process involves intracellular signalling molecules. L-Selectin capping seems to be independent on activation of p56lck-kinase, but requires the neutral sphingomyelinase, small G proteins and the cytoskeleton. Therefore, capping of L-selectin upon stimulation might play an important role in the very early phase of lymphocyte trafficking.

The identification of a volume-regulated anion channel: an amazing Odyssey.

The volume-regulated anion channel (VRAC) plays a pivotal role in cell volume regulation in essentially all cell types studied. Additionally, VRAC appears to contribute importantly to a wide range of other cellular functions and pathological events, including cell motility, cell proliferation, apoptosis and excitotoxic glutamate release in stroke. Although biophysically, pharmacologically and functionally thoroughly described, VRAC has until very recently remained a genetic orphan. The search for the molecular identity of VRAC has been long and has yielded multiple potential candidates, all of which eventually turned out to have properties not fully compatible with those of VRAC. Recently, two groups have independently identified the protein leucine-rich repeats containing 8A (LRRC8A), belonging to family of proteins (LRRC8A-E) distantly related to pannexins, as the likely pore-forming subunit of VRAC. In this brief review, we summarize the history of the discovery of VRAC, outline its basic biophysical and pharmacological properties, link these to several cellular functions in which VRAC appears to play important roles, and sketch the amazing search for the molecular identity of this channel. Finally, we describe properties of the LRRC8 proteins, highlight some features of the LRRC8A knockout mouse and discuss the impact of the discovery of LRRC8 as VRAC on future research.