The ATP-Releasing Maxi-Cl Channel: Its Identity, Molecular Partners and Physiological/Pathophysiological Implications.

The Maxi-Cl phenotype accounts for the majority (app. 60%) of reports on the large-conductance maxi-anion channels (MACs) and has been detected in almost every type of cell, including placenta, endothelium, lymphocyte, cardiac myocyte, neuron, and glial cells, and in cells originating from humans to frogs. A unitary conductance of 300-400 pS, linear current-to-voltage relationship, relatively high anion-to-cation selectivity, bell-shaped voltage dependency, and sensitivity to extracellular gadolinium are biophysical and pharmacological hallmarks of the Maxi-Cl channel. Its identification as a complex with SLCO2A1 as a core pore-forming component and two auxiliary regulatory proteins, annexin A2 and S100A10 (p11), explains the activation mechanism as Tyr23 dephosphorylation at ANXA2 in parallel with calcium binding at S100A10. In the resting state, SLCO2A1 functions as a prostaglandin transporter whereas upon activation it turns to an anion channel. As an efficient pathway for chloride, Maxi-Cl is implicated in a number of physiologically and pathophysiologically important processes, such as cell volume regulation, fluid secretion, apoptosis, and charge transfer. Maxi-Cl is permeable for ATP and other small signaling molecules serving as an electrogenic pathway in cell-to-cell signal transduction. Mutations at the SLCO2A1 gene cause inherited bone and gut pathologies and malignancies, signifying the Maxi-Cl channel as a perspective pharmacological target.

Lytic and sublytic effects of gossypol on red blood cells and thymocytes.

Gossypol is a natural polyphenol presently considered as a promising biological phytochemical with a range of activities including anticancer. We examined volume regulation-dependent effects of gossypol using erythrocytes and thymic lymphocytes. Gossypol effectively lysed human red blood cells (RBC) with a half-maximal concentration of 67.4 ± 1.6 µmol/L and in a non-colloid osmotic manner. Sublytic gossypol doses of 1-10 µmol/L significantly protected RBC from osmotic hemolysis, but potentiated their sensitivity to the colloid-osmotic lysis induced by a pore-former nystatin. When added to the thymocytes suspension, gossypol caused a strong depression of the ability of cells to restore their volume under hypoosmotic stress with a half-maximal activity at 2.1 ± 0.3 µmol/L. Gossypol suppressed regulatory volume decrease under experimental conditions, when cationic permeability was controlled by gramicidin D, and volume recovery depended mainly on anionic conductance, suggesting that the polyphenol inhibits the swelling-induced anion permeability. In direct patch-clamp experiments, gossypol inhibited the volume-sensitive outwardly rectifying (VSOR) chloride channel in thymocytes and in human HCT116 and HeLa cells, possibly by a mechanism when gossypol molecule with a radius close to the size of channel pore plugs into the narrowest portion of the native VSOR chloride channel. Micromolar gossypol suppressed proliferation of thymocytes, HCT116 and HeLa cells. VSOR blockage may represent new mechanism of anticancer activity of gossypol in addition to its action as a BH3-mimetic.

Properties, Structures, and Physiological Roles of Three Types of Anion Channels Molecularly Identified in the 2010's.

Molecular identification was, at last, successfully accomplished for three types of anion channels that are all implicated in cell volume regulation/dysregulation. LRRC8A plus LRRC8C/D/E, SLCO2A1, and TMEM206 were shown to be the core or pore-forming molecules of the volume-sensitive outwardly rectifying anion channel (VSOR) also called the volume-regulated anion channel (VRAC), the large-conductance maxi-anion channel (Maxi-Cl), and the acid-sensitive outwardly rectifying anion channel (ASOR) also called the proton-activated anion channel (PAC) in 2014, 2017, and 2019, respectively. More recently in 2020 and 2021, we have identified the S100A10-annexin A2 complex and TRPM7 as the regulatory proteins for Maxi-Cl and VSOR/VRAC, respectively. In this review article, we summarize their biophysical and structural properties as well as their physiological roles by comparing with each other on the basis of their molecular insights. We also point out unsolved important issues to be elucidated soon in the future.

Annexin A2-S100A10 Represents the Regulatory Component of Maxi-Cl Channel Dependent on Protein Tyrosine Dephosphorylation and Intracellular Ca²âº.

BACKGROUND/AIMS: Maxi-anion channel (Maxi-Cl) is ubiquitously expressed and involved in a number of important cell functions especially by serving as an ATP release pathway. We recently identified SLCO2A1 as its essential core component. However, the regulatory component required for the channel activation/inactivation remains unidentified. METHODS: In the present study, to identify the regulatory component, we made genome-wide analysis combined with siRNA screening and performed patch-clamp studies and ATP release assay after gene silencing and overexpression. RESULTS: Comparative microarray analysis between Maxi-Cl-rich C127 and -deficient C1300 cells revealed highly differential expression not only of SLCO2A1 but also of four annexin family members. Gene silencing study showed that Anxa2 is involved in Maxi-Cl activity. The Maxi-Cl events appeared in C1300 cells by overexpression of Slco2a1 and more efficiently by that of Slco2a1 plus Anxa2. Immunoprecipitation assay supported the interaction between ANXA2 and SLCO2A1. Suppressive effects of overexpression of a phospho-mimicking mutant of Anxa2, Anxa2-Y23E, indicated that protein tyrosine dephosphorylation dependence of Maxi-Cl is conferred by ANXA2. Maxi-Cl activity was suppressed by gene silencing of S100A10, a binding partner of ANXA2, and by applying a synthetic ANXA2 peptide, Ac-(1-14), which interferes with the ANXA2-S100A10 complex formation. Intracellular Ca2+ dependence of Maxi-Cl activity was abolished by S100a10 knockdown. CONCLUSION: The ANXA2-S100A10 complex represents the regulatory component of Maxi-Cl conferring protein tyrosine dephosphorylation dependence and intracellular Ca2+ sensitivity on this channel.

Effect of plant flavonoids on the volume regulation of rat thymocytes under hypoosmotic stress.

BACKGROUND: Cell volume regulation and volume-regulated anion channels are critical for cell survival in non-isosmotic conditions, and dysregulation of this system is detrimental. Although genes and proteins underlying this basic cellular machinery were recently identified, the pharmacology remains poorly explored. METHODS: We examined effects of 16 flavonoids on the regulatory volume decrease (RVD) of thymocytes under hypoosmotic stress assessed by light transmittance and on the activity of volume-sensitive chloride channel by patch-clamp technique. RESULTS: Comparison of effects of flavonoids on RVD revealed a group of four active substances with lehmannin being the strongest inhibitor (IC50 = 8.8 µM). Structure-functional comparison suggested that hydrophobicity brought about by methoxy, prenyl or lavandulyl groups as well as by the absence of glucosyl fragment together with localization of the phenyl ring B at the position C2 (which is at C3 in totally inactive isoflavones) are important structural determinants for the flavonoids activity as volume regulation inhibitors. All active flavonoids suppressed RVD under Gramicidin D-NMDG hypotonic stress conditions when cationic permeability was increased by an ionophore, gramicidin D, with all extracellular monovalent cations replaced with bulky NMDG+ suggesting that they target volume-sensitive anionic permeability. While effects of hispidulin and pulicarin were only partial, lehmannin and pinocembrin completely abolished RVD under Gramicidin D-NMDG conditions. In direct patch-clamp experiments, lehmannin and pinocembrin produced a strong inhibiting effect on the swelling-induced whole-cell chloride conductance in a voltage-independent manner. CONCLUSION: Lehmannin, pinocembrin, and possibly hispidulin and pulicarin may serve as leads for developing effective low-toxic immunomodulators.

The organic anion transporter SLCO2A1 constitutes the core component of the Maxi-Cl channel.

The maxi-anion channels (MACs) are expressed in cells from mammals to amphibians with ~60% exhibiting a phenotype called Maxi-Cl. Maxi-Cl serves as the most efficient pathway for regulated fluxes of inorganic and organic anions including ATP However, its molecular entity has long been elusive. By subjecting proteins isolated from bleb membranes rich in Maxi-Cl activity to LC-MS/MS combined with targeted siRNA screening, CRISPR/Cas9-mediated knockout, and heterologous overexpression, we identified the organic anion transporter SLCO2A1, known as a prostaglandin transporter (PGT), as a key component of Maxi-Cl. Recombinant SLCO2A1 exhibited Maxi-Cl activity in reconstituted proteoliposomes. When SLCO2A1, but not its two disease-causing mutants, was heterologously expressed in cells which lack endogenous SLCO2A1 expression and Maxi-Cl activity, Maxi-Cl currents became activated. The charge-neutralized mutant became weakly cation-selective with exhibiting a smaller single-channel conductance. Slco2a1 silencing in vitro and in vivo, respectively, suppressed the release of ATP from swollen C127 cells and from Langendorff-perfused mouse hearts subjected to ischemia-reperfusion. These findings indicate that SLCO2A1 is an essential core component of the ATP-conductive Maxi-Cl channel.

The properties, functions, and pathophysiology of maxi-anion channels.

The maxi-anion channels (MACs) with a unitary conductance of 200-500 pS are detected in virtually every part of the whole body and found in cells from mammals to amphibia. The channels are normally silent but can be activated by physiologically/pathophysiologically relevant stimuli, such as osmotic, salt, metabolic, oxidative, and mechanical stresses, receptor activation, serum, heat, and intracellular Ca(2+) rise. In some MACs, protein dephosphorylation is associated with channel activation. Among MACs so far studied, around 60 % (designated here as Maxi-Cl) possess, in common, the following phenotypical biophysical properties: (1) unitary conductance of 300-400 pS, (2) a linear current-voltage relationship, (3) high anion-to-cation selectivity with PCl/Pcation of >8, and (4) inactivation at positive and negative potentials over a certain level (usually ±20 mV). The pore configuration of the Maxi-Cl is asymmetrical with extracellular and intracellular radii of ∼1.42 and ∼1.16 nm, respectively, and a medial constriction down to ∼0.55-0.75 nm. The classical function of MACs is control of membrane potential and fluid movement. Permeability to ATP and glutamate turns MACs to signaling channels in purinergic and glutamatergic signal transduction defining them as a perspective target for drug discovery. The molecular identification is an urgent task that would greatly promote the developments in this field. A possible relationship between these channels and some transporters is discussed.

Plasmalemmal VDAC controversies and maxi-anion channel puzzle.

The maxi-anion channel has been observed in many cell types from the very beginning of the patch-clamp era. The channel is highly conductive for chloride and thus can modulate the resting membrane potential and play a role in fluid secretion/absorption and cell volume regulation. A wide nanoscopic pore of the maxi-anion channel permits passage of excitatory amino acids and nucleotides. The channel-mediated release of these signaling molecules is associated with kidney tubuloglomerular feedback, cardiac ischemia/hypoxia, as well as brain ischemia/hypoxia and excitotoxic neurodegeneration. Despite the ubiquitous expression and physiological/pathophysiological significance, the molecular identity of the maxi-anion channel is still obscure. VDAC is primarily a mitochondrial protein; however several groups detected it on the cellular surface. VDAC in lipid bilayers reproduced the most important biophysical properties of the maxi-anion channel, such as a wide nano-sized pore, closure in response to moderately high voltages, ATP-block and ATP-permeability. However, these similarities turned out to be superficial, and the hypothesis of plasmalemmal VDAC as the maxi-anion channel did not withstand the test by genetic manipulations of VDAC protein expression. VDAC on the cellular surface could also function as a ferricyanide reductase or a receptor for plasminogen kringle 5 and for neuroactive steroids. These ideas, as well as the very presence of VDAC on plasmalemma, remain to be scrutinized by genetic manipulations of the VDAC protein expression. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

Interaction of heparins and dextran sulfates with a mesoscopic protein nanopore.

A mechanism of how polyanions influence the channel formed by Staphylococcus aureus alpha-hemolysin is described. We demonstrate that the probability of several types of polyanions to block the ion channel depends on the presence of divalent cations and the polyanion molecular weight and concentration. For heparins, a 10-fold increase in molecular weight decreases the half-maximal inhibitory concentration, IC(50), nearly 10(4)-fold. Dextran sulfates were less effective at blocking the channel. The polyanions are significantly more effective at reducing the conductance when added to the trans side of this channel. Lastly, the effectiveness of heparins on the channel conductance correlated with their influence on the zeta-potential of liposomes. A model that includes the binding of polyanions to the channel-membrane complex via Ca(2+)-bridges and the asymmetry of the channel structure describes the data adequately. Analysis of the single channel current noise of wild-type and site-directed mutant versions of alpha-hemolysin channels suggests that a single polyanion enters the pore due to electrostatic forces and physically blocks the ion conduction path. The results might be of interest for pharmacology, biomedicine, and research aiming to design mesoscopic pore blockers.

Pore formation by Vibrio cholerae cytolysin requires cholesterol in both monolayers of the target membrane.

Vibrio cholerae cytolysin (VCC) forms oligomeric transmembrane pores in cholesterol-rich membranes. To better understand this process, we used planar bilayer membranes. In symmetric membranes, the rate of the channel formation by VCC has a superlinear dependency on the cholesterol membrane fraction. Thus, more than one cholesterol molecule can facilitate VCC-pore formation. In asymmetric membranes, the rate of pore formation is limited by the leaflet with the lower cholesterol content. Methyl-beta-cyclodextrin, which removes cholesterol from membranes, rapidly inhibits VCC pore formation, even when it is added to the side opposite that of VCC addition. The results suggest that cholesterol in both membrane leaflets aid VCC-pore formation and that either leaflet can function as a kinetic bottleneck with respect to the rate of pore-formation.

Conductance and ion selectivity of a mesoscopic protein nanopore probed with cysteine scanning mutagenesis.

Nanometer-scale proteinaceous pores are the basis of ion and macromolecular transport in cells and organelles. Recent studies suggest that ion channels and synthetic nanopores may prove useful in biotechnological applications. To better understand the structure-function relationship of nanopores, we are studying the ion-conducting properties of channels formed by wild-type and genetically engineered versions of Staphylococcus aureus alpha-hemolysin (alphaHL) reconstituted into planar lipid bilayer membranes. Specifically, we measured the ion selectivities and current-voltage relationships of channels formed with 24 different alphaHL point cysteine mutants before and after derivatizing the cysteines with positively and negatively charged sulfhydryl-specific reagents. Novel negative charges convert the selectivity of the channel from weakly anionic to strongly cationic, and new positive charges increase the anionic selectivity. However, the extent of these changes depends on the channel radius at the position of the novel charge (predominantly affects ion selectivity) or on the location of these charges along the longitudinal axis of the channel (mainly alters the conductance-voltage curve). The results suggest that the net charge of the pore wall is responsible for cation-anion selectivity of the alphaHL channel and that the charge at the pore entrances is the main factor that determines the shape of the conductance-voltage curves.

Protein electrostriction: a possibility of elastic deformation of the alpha-hemolysin channel by the applied field.

While conformational flexibility of proteins is widely recognized as one of their functionally crucial features and enjoys proper attention for this reason, their elastic properties are rarely discussed. In ion channel studies, where the voltage-induced or ligand-induced conformational transitions, gating, are the leading topic of research, the elastic structural deformation by the applied electric field has never been addressed at all. Here we examine elasticity using a model channel of known crystal structure-Staphylococcus aureus alpha-hemolysin. Working with single channels reconstituted into planar lipid bilayers, we first show that their ionic conductance is asymmetric with voltage even at the highest salt concentration used where the static charges in the channel interior are maximally shielded. Second, choosing 18-crown-6 as a molecular probe whose size is close to the size of the narrowest part of the alpha-hemolysin pore, we analyze the blockage of the channel by the crown/K(+) complex. Analysis of the blockage within the framework of the Woodhull model in its generalized form demonstrates that the model is able to correctly describe the crown effect only if the parameters of the model are considered to be voltage-dependent. Specifically, one has to include either a voltage-dependent barrier for crown release to the cis side of the channel or voltage-dependent interactions between the binding site and the crown. We suggest that the voltage sensitivity of both the ionic conductance of the channel seen at the highest salt concentration and its blockage by the crown reflects a field-induced deformation of the pore.

Probing the volume changes during voltage gating of Porin 31BM channel with nonelectrolyte polymers.

To probe the volume changes of the voltage-dependent anion-selective channel (VDAC), the nonelectrolyte exclusion technique was taken because it is one of the few existing methods that may define quite accurately the rough geometry of lumen of ion channels (in membranes) for which there is no structural data.Here, we corroborate the data from our previous study [FEBS Lett. 416 (1997) 187] that the gross structural features of VDAC in its highest conductance state are asymmetric with respect to the plane of the membrane, and state that this asymmetry is not dependent on sign of voltage applied. Hence, the plasticity of VDAC does not play a role in the determination of lumen geometry at this state and the asymmetry is an internal property of the channel. We also show that the apparent diameter of the cis segment of the pore decreases slightly from 2 to 1.8 nm when the channel's conductance decreases from its high to low state. However, the trans funnel segment undergoes a more marked change in polymer accessible volume. Specifically, its larger diameter decreases from approximately 4 to 2.4 nm. Supposing the channel's total length is 4.6 nm, the apparent change in channel volume during this transition is estimated to be about 10 nm(3), i.e. about 40% of the channel's volume in the high conductance state.