Purification, Reconstitution, and Functional Analysis of Connexin Hemichannels.

Connexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin isoform, can increase hemichannel activity and cause deafness. Studies using purified isolated systems under well-controlled conditions are essential for a full understanding of molecular mechanisms of hemichannel function under normal conditions and in disease, and here, we present methodology for the expression, purification, and functional analysis of hemichannels formed by Cx26.

Extracellular Cysteines Are Critical to Form Functional Cx46 Hemichannels.

Connexin (Cxs) hemichannels participate in several physiological and pathological processes, but the molecular mechanisms that control their gating remain elusive. We aimed at determining the role of extracellular cysteines (Cys) in the gating and function of Cx46 hemichannels. We studied Cx46 and mutated all of its extracellular Cys to alanine (Ala) (one at a time) and studied the effects of the Cys mutations on Cx46 expression, localization, and hemichannel activity. Wild-type Cx46 and Cys mutants were expressed at comparable levels, with similar cellular localization. However, functional experiments showed that hemichannels formed by the Cys mutants did not open either in response to membrane depolarization or removal of extracellular divalent cations. Molecular-dynamics simulations showed that Cys mutants may show a possible alteration in the electrostatic potential of the hemichannel pore and an altered disposition of important residues that could contribute to the selectivity and voltage dependency in the hemichannels. Replacement of extracellular Cys resulted in "permanently closed hemichannels", which is congruent with the inhibition of the Cx46 hemichannel by lipid peroxides, through the oxidation of extracellular Cys. These results point to the modification of extracellular Cys as potential targets for the treatment of Cx46-hemichannel associated pathologies, such as cataracts and cancer, and may shed light into the gating mechanisms of other Cx hemichannels.

Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala.

Astrocytes release gliotransmitters via connexin 43 (Cx43) hemichannels into neighboring synapses, which can modulate synaptic activity and are necessary for fear memory consolidation. However, the gliotransmitters released, and their mechanisms of action remain elusive. Here, we report that fear conditioning training elevated Cx43 hemichannel activity in astrocytes from the basolateral amygdala (BLA). The selective blockade of Cx43 hemichannels by microinfusion of TAT-Cx43L2 peptide into the BLA induced memory deficits 1 and 24 h after training, without affecting learning. The memory impairments were prevented by the co-injection of glutamate and D-serine, but not by the injection of either alone, suggesting a role for NMDA receptors (NMDAR). The incubation with TAT-Cx43L2 decreased NMDAR-mediated currents in BLA slices, effect that was also prevented by the addition of glutamate and D-serine. NMDARs in primary neuronal cultures were unaffected by TAT-Cx43L2, ruling out direct effects of the peptide on NMDARs. Finally, we show that D-serine permeates through purified Cx43 hemichannels reconstituted in liposomes. We propose that the release of glutamate and D-serine from astrocytes through Cx43 hemichannels is necessary for the activation of post-synaptic NMDARs during training, to allow for the formation of short-term and subsequent long-term memory, but not for learning per se.

Connexin hemichannel inhibitors with a focus on aminoglycosides.

Connexins are membrane proteins involved directly in cell-to-cell communication through the formation of gap-junctional channels. These channels result from the head-to-head docking of two hemichannels, one from each of two adjacent cells. Undocked hemichannels are also present at the plasma membrane where they mediate the efflux of molecules that participate in autocrine and paracrine signaling, but abnormal increase in hemichannel activity can lead to cell damage in disorders such as cardiac infarct, stroke, deafness, cataracts, and skin diseases. For this reason, connexin hemichannels have emerged as a valid therapeutic target. Know small molecule hemichannel inhibitors are not ideal leads for the development of better drugs for clinical use because they are not specific and/or have toxic effects. Newer inhibitors are more selective and include connexin mimetic peptides, anti-connexin antibodies and drugs that reduce connexin expression such as antisense oligonucleotides. Re-purposed drugs and their derivatives are also promising because of the significant experience with their clinical use. Among these, aminoglycoside antibiotics have been identified as inhibitors of connexin hemichannels that do not inhibit gap-junctional channels. In this review, we discuss connexin hemichannels and their inhibitors, with a focus on aminoglycoside antibiotics and derivatives of kanamycin A that inhibit connexin hemichannels, but do not have antibiotic effect.

Polymer-Encased Nanodiscs and Polymer Nanodiscs: New Platforms for Membrane Protein Research and Applications.

Membrane proteins (MPs) are essential to many organisms' major functions. They are notorious for being difficult to isolate and study, and mimicking native conditions for studies in vitro has proved to be a challenge. Lipid nanodiscs are among the most promising platforms for MP reconstitution, but they contain a relatively labile lipid bilayer and their use requires previous protein solubilization in detergent. These limitations have led to the testing of copolymers in new types of nanodisc platforms. Polymer-encased nanodiscs and polymer nanodiscs support functional MPs and address some of the limitations present in other MP reconstitution platforms. In this review, we provide a summary of recent developments in the use of polymers in nanodiscs.

Amphiphilic aminoglycosides with increased selectivity for inhibition of connexin 43 (Cx43) hemichannels.

Gap junction channels formed by the association of connexin hemichannels play a crucial role in intercellular communication. Connexin 43 (Cx43) is expressed in a variety of tissues and organs, including heart and brain, and abnormal sustained opening of undocked "free" hemichannels contributes to the cell damage in cardiac infarcts and stroke. Selective inhibitors of Cx43 hemichannels for clinical use are then desirable. Here, we synthesized and tested new aminoglycosides for their connexin inhibitory activity towards Cx26 and Cx43 hemichannels. The lead compounds displayed enhanced Cx43/Cx26 selectivity for hemichannel inhibition when compared to the parent kanamycin A and other commercially available aminoglycosides. These lead compounds are not cytotoxic to mammalian cells and show promise for the treatment of ischemic damage of the heart, brain, and kidneys. We identified a new compound as a promising lead based on its good selectivity for Cx43 hemichannels inhibition and the simplicity and affordability of its production.

Extraction and reconstitution of membrane proteins into lipid nanodiscs encased by zwitterionic styrene-maleic amide copolymers.

Membrane proteins can be reconstituted in polymer-encased nanodiscs for studies under near-physiological conditions and in the absence of detergents, but traditional styrene-maleic acid copolymers used for this purpose suffer severely from buffer incompatibilities. We have recently introduced zwitterionic styrene-maleic amide copolymers (zSMAs) to overcome this limitation. Here, we compared the extraction and reconstitution of membrane proteins into lipid nanodiscs by a series of zSMAs with different styrene:maleic amide molar ratios, chain sizes, and molecular weight distributions. These copolymers solubilize, stabilize, and support membrane proteins in nanodiscs with different efficiencies depending on both the structure of the copolymers and the membrane proteins.

4-Hydroxynonenal induces Cx46 hemichannel inhibition through its carbonylation.

Hemichannels formed by connexins mediate the exchange of ions and signaling molecules between the cytoplasm and the extracellular milieu. Under physiological conditions hemichannels have a low open probability, but in certain pathologies their open probability increases, which can result in cell damage. Pathological conditions are characterized by the production of a number of proinflammatory molecules, including 4-hydroxynonenal (4-HNE), one of the most common lipid peroxides produced in response to inflammation and oxidative stress. The aim of this work was to evaluate whether 4-HNE modulates the activity of Cx46 hemichannels. We found that 4-HNE (100 µM) reduced the rate of 4',6-diamino-2-fenilindol (DAPI) uptake through hemichannels formed by recombinant human Cx46 fused to green fluorescent protein, an inhibition that was reversed partially by 10 mM dithiothreitol. Immunoblot analysis showed that the recombinant Cx46 expressed in HeLa cells becomes carbonylated after exposure to 4-HNE, and that 10 mM dithiothreitol reduced its carbonylation. We also found that Cx46 was carbonylated by 4-HNE in the lens of a selenite-induced cataract animal model. The exposure to 100 µM 4-HNE decreased hemichannel currents formed by recombinant rat Cx46 in Xenopus laevis oocytes. This inhibition also occurred in a mutant expressing only the extracellular loop cysteines, suggesting that other Cys are not responsible for the hemichannel inhibition by carbonylation. This work demonstrates for the first time that Cx46 is post-translationally modified by a lipid peroxide and that this modification reduces Cx46 hemichannel activity.

A Simple Assay to Evaluate the Function of Human Connexin Hemichannels Expressed in Escherichia coli that Can Be Used for Drug Discovery and Mutant Analysis.

Abnormally increased activity of connexin hemichannels contributes to cell damage in many disorders, including deafness, stroke, and cardiac infarct, and therefore hemichannels constitute a potentially important therapeutic target. Unfortunately, the available hemichannel inhibitors are not specific and most are toxic. The absence of a simple and cost-effective screening assay has made the discovery of hemichannel inhibitors difficult. Here, we present an optimized assay where human connexins are expressed in genetically modified Escherichia coli cells deficient in potassium uptake (LB2003 cells). These cells cannot grow in low-potassium medium, and hemichannel function is assayed by the reversion of the no-growth phenotype. Since functional hemichannels are permeable to potassium, they allow for its uptake and cell growth. The simple reading of bacterial growth in low-potassium medium distinguishes functional hemichannels (growth) from those inhibited (no growth). This assay is simple, robust, inexpensive, and reliable, and is easily scaled to high-throughput multiwell platforms. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of competent LB2003 cells resistant to kanamycin Basic Protocol 2: Growth complementation assay Support Protocol: Evaluation of cytotoxic effects of potential connexin hemichannel inhibitors.

Functional and structural comparison of the ABC exporter MsbA studied in detergent and reconstituted in nanodiscs.

Purified membrane proteins are most frequently studied solubilized in detergent, but the properties of detergent micelles are very different from those of lipid bilayers. Therefore, there is an increasing interest in studying membrane proteins under conditions that resemble the membrane protein native environment more closely. Although there are indications of differences between membrane proteins in detergent and in lipid bilayers, direct functional and structural comparisons are very hard to find. Nanodiscs have been established as a new platform that consists of two molecules of a membrane scaffold protein that surround a small lipid-bilayer patch. Here, we undertook the task of comparing the function and conformational states of the transport protein MsbA in detergent and nanodiscs using ATPase activity and luminescence resonance energy transfer (LRET) measurements to assess differences in activity and conformational states, respectively. MsbA is a prototypical member of the ATP binding cassette protein superfamily. MsbA activity was higher in nanodiscs vs detergent, which had clear structural correlates: an increase in the fraction of molecules displaying closed nucleotide-binding domain dimers in the apo state, and a decrease in the distance of the "dissociated" nucleotide-binding domains. Our LRET studies support the notion that the widely separated nucleotide binding domains observed in the MsbA x-ray structures in detergent do not correspond to physiological conformations. Although our studies focus on a particular ABC exporter, the possibility of similar environment effects on other membrane proteins should be carefully considered.

Inhibition of Connexin Hemichannels by New Amphiphilic Aminoglycosides without Antibiotic Activity.

Connexins hemichannels (HCs) from adjacent cells form gap junctional channels that mediate cell-to-cell communication. Abnormal opening of "free" undocked HCs can produce cell damage and participate in the mechanism of disorders such as cardiac infarct, stroke, deafness, skin diseases, and cataracts. Therefore, inhibitors of connexin HCs have great pharmacological potential. Antibiotic aminoglycosides (AGs) have been recently identified as connexin HC inhibitors, but their antibiotic effect is an issue for the treatment of disorders where infections do not play a role. Herein, we synthesized and tested several amphiphilic AGs without antibiotic effect for their inhibition against connexin HCs, using a newly developed cell-based bacterial growth complementation assay. Several leads with superior potency than the parent compound, kanamycin A, were identified. Unlike traditional AGs, these amphiphilic AGs are not bactericidal and are not toxic to mammalian cells, making them better than traditional AGs as HC inhibitors for clinical use and other applications.

Inhibition by Commercial Aminoglycosides of Human Connexin Hemichannels Expressed in Bacteria.

In addition to gap junctional channels that mediate cell-to-cell communication, connexins form hemichannels that are present at the plasma membrane. Since hemichannels are permeable to small hydrophilic compounds, including metabolites and signaling molecules, their abnormal opening can cause or contribute to cell damage in disorders such as cardiac infarct, stroke, deafness, skin diseases, and cataracts. Therefore, hemichannels are potential pharmacological targets. A few aminoglycosides, well-known broad-spectrum antibiotics, have been shown to inhibit hemichannels. Here, we tested several commercially available aminoglycosides for inhibition of human connexin hemichannels using a cell-based bacterial growth complementation assay that we developed recently. We found that kanamycin A, kanamycin B, geneticin, neomycin, and paromomycin are effective inhibitors of hemichannels formed by connexins 26, 43, and 46 (Cx26, Cx43, and Cx46). Because of the >70 years of clinical experience with aminoglycosides and the fact that several of the aminoglycosides tested here have been used in humans, they are promising starting points for the development of effective connexin hemichannel inhibitors.

Polymer Nanodiscs: Discoidal Amphiphilic Block Copolymer Membranes as a New Platform for Membrane Proteins.

Lipid nanodiscs are playing increasingly important roles in studies of the structure and function of membrane proteins. Development of lipid nanodiscs as a membrane-protein-supporting platform, or a drug targeting and delivery vehicle in general, is undermined by the fluidic and labile nature of lipid bilayers. Here, we report the discovery of polymer nanodiscs, i.e., discoidal amphiphilic block copolymer membrane patches encased within membrane scaffold proteins, as a novel two-dimensional nanomembrane that maintains the advantages of lipid nanodiscs while addressing their weaknesses. Using MsbA, a bacterial ATP-binding cassette transporter as a membrane protein prototype, we show that the protein can be reconstituted into the polymer nanodiscs in an active state. As with lipid nanodiscs, reconstitution of detergent-solubilized MsbA into the polymer nanodiscs significantly enhances its activity. In contrast to lipid nanodiscs that undergo time- and temperature-dependent structural changes, the polymer nanodiscs experience negligible structural evolution under similar environmental stresses, revealing a critically important property for the development of nanodisc-based characterization methodologies or biotechnologies. We expect that the higher mechanical and chemical stability of block copolymer membranes and their chemical versatility for adaptation will open new opportunities for applications built upon diverse membrane protein functions, or involved with drug targeting and delivery.

Polymer-encased nanodiscs with improved buffer compatibility.

Styrene-maleic acid copolymers allow for solubilization and reconstitution of membrane proteins into nanodiscs. These polymer-encased nanodiscs are promising platforms for studies of membrane proteins in a near-physiologic environment without the use of detergents. However, current styrene-maleic acid copolymers display severe limitations in terms of buffer compatibility and ensued flexibility for various applications. Here, we present a new family of styrene-maleic acid copolymers that do not aggregate at low pH or in the presence of polyvalent cations, and can be used to solubilize membrane proteins and produce nanodiscs of controlled sizes.

Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter (PCFT, SLC46A1).

The proton-coupled folate transporter (PCFT) provides an essential uptake route for the vitamin folic acid (B9) in mammals. In addition, it is currently of high interest for targeting chemotherapeutic agents to tumors due to the increased folic acid requirement of rapidly dividing tumor cells as well as the upregulated PCFT expression in several tumors. To understand its function, determination of its atomic structure and molecular mechanism of transport are essential goals that require large amounts of functional PCFT. Here, we present a high-level heterologous expression system for human PCFT using a recombinant baculovirus and Spodoptera frugiperda (Sf9) insect cells. We demonstrate folate transport functionality along the PCFT expression, isolation, and purification process. Importantly, purified PCFT transports folic acid after reconstitution. We thus succeeded in overcoming heterologous expression as a major bottleneck of PCFT research. The availability of an overexpression system for human PCFT provides the basis for future biochemical, biophysical and structural studies.

A Cell-Based Assay to Assess Hemichannel Function.

Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an attractive therapeutic target. Unfortunately, most available inhibitors are not selective or isoform specific, which hampers their translational application. The absence of a battery of useful inhibitors is due in part to the absence of simple screening assays for the discovery of hemichannel-active drugs. Here, we present an assay that we have recently developed to assess hemichannel function. The assay is based on the expression of functional human connexins in a genetically modified bacterial strain deficient in K+ uptake. These modified cells do not grow in low-K+ medium, but functional expression of connexin hemichannels allows K+ uptake and growth. This cell-growth-based assay is simple, robust, and easily scalable to high-throughput multi-well platforms.

An Escherichia coli-Based Assay to Assess the Function of Recombinant Human Hemichannels.

Connexins form the gap junctional channels that mediate cell-to-cell communication, and also form hemichannels present at the plasma membrane. Hemichannels are permeable to small hydrophilic compounds, including molecules involved in autocrine and paracrine signaling. An abnormal hemichannel opening causes or contributes to cell damage in common human disorders (e.g., cardiac infarct, cerebrovascular accidents, deafness, skin diseases, and cataracts) and is therefore a potential pharmacological target. The discovery of useful hemichannels inhibitors has been hampered in part by the lack of suitable high-throughput functional assays. Here, we developed and characterized an assay useful to assess the function of hemichannels formed by human connexins expressed in a genetically modified Escherichia coli strain. The LB2003 cells, devoid of three key K+ uptake transport mechanisms, cannot grow in low-[K+] medium, but expression of Cx26, Cx43, or Cx46 rescues their growth defect (growth complementation). We developed a protocol for a simple, inexpensive, easily scalable, reproducible, and sensitive assay that should be useful for the discovery of new and better hemichannel inhibitors based on the analysis of small-compound libraries.

Gap-junctional channel and hemichannel activity of two recently identified connexin 26 mutants associated with deafness.

Gap-junction channels (GJCs) are formed by head-to-head association of two hemichannels (HCs, connexin hexamers). HCs and GJCs are permeable to ions and hydrophilic molecules of up to Mr ~1 kDa. Hearing impairment of genetic origin is common, and mutations of connexin 26 (Cx26) are its major cause. We recently identified two novel Cx26 mutations in hearing-impaired subjects, L10P and G109V. L10P forms functional GJCs with slightly altered voltage dependence and HCs with decrease ATP/cationic dye selectivity. G109V does not form functional GJCs, but forms functional HCs with enhanced extracellular Ca(2+) sensitivity and subtle alterations in voltage dependence and ATP/cationic dye selectivity. Deafness associated with G109V could result from decreased GJCs activity, whereas deafness associated to L10P may have a more complex mechanism that involves changes in HC permeability.

Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors.

Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs.

Functional hemichannels formed by human connexin 26 expressed in bacteria.

Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure-function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs.