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Although the majority of eukaryotic MEMBRANE PROTEIN structures are DERIVED FROM PROTEINS produced in HEK293 and insect cells, the authors show here the importance of yeast as a production host and its role as an essential player in the production of eukaryotic membrane proteins for structural and functional analysis. Membrane proteins are challenging targets to functionally and structurally characterize. An enduring bottleneck in their study is the reliable production of sufficient yields of stable protein. Here, we evaluate all eukaryotic membrane protein production experiments that have supported the deposition of a high-resolution structure. We focused on the most common yeast host systems, Saccharomyces cerevisiae and Pichia pastoris. The first high-resolution structure of a membrane protein produced in yeast was described in 1999 and today there are 186 structures of alpha-helical membrane proteins, representing 101 unique proteins from 37 families. Homologous and heterologous production are equally common in S. cerevisiae, while heterologous production dominates in P. pastoris, especially of human proteins, which represent about one-third of the total. Investigating protein engineering approaches (78 proteins from seven families) demonstrated that the majority contained a polyhistidine tag for purification, typically at the C-terminus of the protein. Codon optimization and truncation of hydrophilic extensions were also common approaches to improve yields. We conclude that yeast remains a useful production host for the study of alpha-helical membrane proteins.

Membrane proteins are an essential part of the machinery of life. They connect the interior and exterior of cells, play an important role in cell signaling and are responsible for the influx and efflux of nutrients and metabolites. For their structural and functional analysis high yields of correctly folded and modified protein are needed. Insect cells, such as Sf9 cells, have been one of the major expression hosts for eukaryotic membrane proteins in structural investigations during the last decade, as they are easier to handle than mammalian cells and provide more natural posttranslational modifications than microbial systems. Here we describe general techniques for establishing and maintaining insect cell cultures, the generation and amplification of recombinant baculovirus stocks using the flashBAC™ or Bac-to-Bac™ systems, membrane protein production, as well as the production of membrane preparations for extraction and purification experiments.

One of the big challenges for the study of structure and function of membrane proteins is the need to extract them from the membrane. Traditionally this was achieved using detergents which disrupt the membrane and form a micelle around the protein, but this can cause issues with protein function and/or stability. In 2009 an alternative approach was reported, using styrene maleic acid (SMA) copolymer to extract small discs of lipid bilayer encapsulated by the polymer and termed SMALPs (SMA lipid particles). Since then this approach has been shown to work for a range of different proteins from many different expression systems. It allows the extraction and purification of a target protein while maintaining a lipid bilayer environment. Recently this has led to several new high-resolution structures and novel insights to function. As with any method there are some limitations and issues to be aware of. Here we describe a standard protocol for preparation of the polymer and its use for membrane protein purification, and also include details of typical challenges that may be encountered and possible ways to address those.

Over the decades, the bacterium Escherichia coli (E. coli) has become the cornerstone of recombinant protein production, used for heterologous synthesis of a variety of membrane proteins. Due to its rapid growth to high densities in cheap media, and its ease of manipulation and handling, E. coli is an excellent host cell for a range of membrane protein targets. Furthermore, its genetic tractability allows for a variety of gene constructs to be screened for optimal expression conditions, resulting in relatively high yields of membrane protein in a short amount of time. Here, we describe the general workflow for the production of membrane proteins in E. coli. The protocols we provide show how the gene of interest is modified, transferred to an expression vector and host, and how membrane protein yields can be optimized and analyzed. The examples we illustrate are well suited for scientists who are starting their journey into the world of membrane protein production.

The aquaporins (AQPs) form a family of integral membrane proteins that facilitate the movement of water across biological membrane by osmosis, as well as facilitating the diffusion of small polar solutes. AQPs have been recognised as drug targets for a variety of disorders associated with disrupted water or solute transport, including brain oedema following stroke or trauma, epilepsy, cancer cell migration and tumour angiogenesis, metabolic disorders, and inflammation. Despite this, drug discovery for AQPs has made little progress due to a lack of reproducible high-throughput assays and difficulties with the druggability of AQP proteins. However, recent studies have suggested that targetting the trafficking of AQP proteins to the plasma membrane is a viable alternative drug target to direct inhibition of the water-conducting pore. Here we review the literature on the trafficking of mammalian AQPs with a view to highlighting potential new drug targets for a variety of conditions associated with disrupted water and solute homeostasis.

Lipases are enzymes of industrial importance responsible for the hydrolysis of ester bonds of triglycerides. A lipolytic fungus was isolated and subsequently identified based on the ITS sequence analysis as putative

The aquaporins (AQPs) are a family of small integral membrane proteins that facilitate the bidirectional transport of water across biological membranes in response to osmotic pressure gradients as well as enable the transmembrane diffusion of small neutral solutes (such as urea, glycerol, and hydrogen peroxide) and ions. AQPs are expressed throughout the human body. Here, we review their key roles in fluid homeostasis, glandular secretions, signal transduction and sensation, barrier function, immunity and inflammation, cell migration, and angiogenesis. Evidence from a wide variety of studies now supports a view of the functions of AQPs being much more complex than simply mediating the passive flow of water across biological membranes. The discovery and development of small-molecule AQP inhibitors for research use and therapeutic development will lead to new insights into the basic biology of and novel treatments for the wide range of AQP-associated disorders.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15539. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate. ispartof: BRITISH JOURNAL OF PHARMACOLOGY vol:178 pages:S157-S245 ispartof: location:England status: published

Aquaporin channels facilitate bidirectional water flow in all cells and tissues. AQP4 is highly expressed in astrocytes. In the CNS, it is enriched in astrocyte endfeet, at synapses, and at the glia limitans, where it mediates water exchange across the blood–spinal cord and blood–brain barriers (BSCB/BBB), and controls cell volume, extracellular space volume, and astrocyte migration. Perivascular enrichment of AQP4 at the BSCB/BBB suggests a role in glymphatic function. Recently, we have demonstrated that AQP4 localization is also dynamically regulated at the subcellular level, affecting membrane water permeability. Ageing, cerebrovascular disease, traumatic CNS injury, and sleep disruption are established and emerging risk factors in developing neurodegeneration, and in animal models of each, impairment of glymphatic function is associated with changes in perivascular AQP4 localization. CNS oedema is caused by passive water influx through AQP4 in response to osmotic imbalances. We have demonstrated that reducing dynamic relocalization of AQP4 to the BSCB/BBB reduces CNS oedema and accelerates functional recovery in rodent models. Given the difficulties in developing pore-blocking AQP4 inhibitors, targeting AQP4 subcellular localization opens up new treatment avenues for CNS oedema, neurovascular and neurodegenerative diseases, and provides a framework to address fundamental questions about water homeostasis in health and disease.

Aquaporins and aquaglyceroporins are membrane channels that allow the permeation of water and certain other small solutes across the cell membrane, or in the case of AQP6, AQP11 and AQP12A, intracellular membranes, such as vesicles and the endoplasmic reticulum membrane [16]. Since the isolation and cloning of the first aquaporin (AQP1) [20], 12 additional mammalian members of the family have been identified, although little is known about the functional properties of one of these (AQP12A; Q8IXF9) and it is thus not tabulated. The other 12 aquaporins can be broadly divided into three families: orthodox aquaporins (AQP0,-1,-2,-4,-5, -6 and -8) permeable mainly to water, but for some additional solutes [4]; aquaglyceroporins (AQP3,-7 -9 and -10), additionally permeable to glycerol and for some isoforms urea [14], and superaquaporins (AQP11 and 12) located within cells [12]. Some aquaporins also conduct ammonia and/or H2O2 giving rise to the terms 'ammoniaporins' ('aquaammoniaporins') and 'peroxiporins', respectively. Aquaporins are impermeable to protons and other inorganic and organic cations, with the possible exception of AQP1, although this is controversial [14]. One or more members of this family of proteins have been found to be expressed in almost all tissues of the body [reviewed in Yang (2017) [26]]. AQPs are involved in numerous processes that include systemic water homeostasis, adipocyte metabolism, brain oedema, cell migration and fluid secretion by epithelia. Loss of function mutations of some human AQPs, or their disruption by autoantibodies further underscore their importance [reviewed by Verkman et al. (2014) [23], Kitchen et al. (2105) [14]]. Functional AQPs exist as homotetramers that are the water conducting units wherein individual AQP subunits (each a protomer) have six TM helices and two half helices that constitute a seventh 'pseudotransmembrane domain' that surrounds a narrow water conducting channel [16]. In addition to the four pores contributed by the protomers, an additional hydrophobic pore exists within the center of the complex [16] that may mediate the transport through AQP1. Although numerous small molecule inhibitors of aquaporins, particularly APQ1, have been reported primarily from Xenopus oocyte swelling assays, the activity of most has subsequently been disputed upon retesting using assays of water transport that are less prone to various artifacts [5] and they are therefore excluded from the tables [see Tradtrantip et al. (2017) [22] for a review].

Lignocellulosic biomass is the most abundant and sustainable feedstock available globally. As a source of the polysaccharides, cellulose and hemicellulose, it can be converted into biofuels and other platform chemicals. This article highlights some important aspects that needs to be focused upon for the commercial development of lignocellulosic biorefineries. Although, lignocellulosic biomass offers clear value in terms of its green advantages and sustainability, there has been very low commercial success at industrial production levels. This can be attributed to a few key factors such as an irregular biomass supply chain, inefficient or complex pre-treatment and saccharification technologies, and scale up challenges leading to high capital and operating expenditures. Moreover, techno-economic studies performed on lignocellulosic biorefineries have revealed that process complexity is the most detrimental factor prohibiting scale-up. Although there have been several research efforts funded both by the public and private sectors, biomass valorization into biofuels and chemicals remains a technical and economical challenge. This review examines the global drivers towards the advancements of lignocellulosic biorefineries, technical and operational challenges for industrialization and future directions towards overcoming them.

Styrene maleic acid (SMA) polymers have proven to be very successful for the extraction of membrane proteins, forming SMA lipid particles (SMALPs), which maintain a lipid bilayer around the membrane protein. SMALP-encapsulated membrane proteins can be used for functional and structural studies. The SMALP approach allows retention of important protein-annular lipid interactions, exerts lateral pressure, and offers greater stability than traditional detergent solubilisation. However, SMA polymer does have some limitations, including a sensitivity to divalent cations and low pH, an absorbance spectrum that overlaps with many proteins, and possible restrictions on protein conformational change. Various modified polymers have been developed to try to overcome these challenges, but no clear solution has been found. A series of partially-esterified variants of SMA (SMA 2625, SMA 1440 and SMA 17352) has previously been shown to be highly effective for solubilisation of plant and cyanobacterial thylakoid membranes. It was hypothesised that the partial esterification of maleic acid groups would increase tolerance to divalent cations. Therefore, these partially-esterified polymers were tested for the solubilisation of lipids and membrane proteins, and their tolerance to magnesium ions. It was found that all partially esterified polymers were capable of solubilising and purifying a range of membrane proteins, but the yield of protein was lower with SMA 1440, and the degree of purity was lower for both SMA 1440 and SMA 17352. SMA 2625 performed comparably to SMA 2000. SMA 1440 also showed an increased sensitivity to divalent cations. Thus, it appears the interactions between SMA and divalent cations are more complex than proposed and require further investigation., Graphical abstract Unlabelled Image, Highlights • Partially-esterified SMA polymers are effective for solubilisation of membrane proteins from different expression systems. • Proteins in partially-esterified SMALPs can be purified by Ni2+ affinity chromatography, but yield is lower with SMA 1440. • Partial esterification of SMA does not overcome the sensitivity to divalent cations. • SMA1440 is more sensitive to magnesium ions than SMA 2000.

Aquaporins are attractive targets for therapeutic intervention in the diverse conditions associated with water and solute dyshomeostasis that affect millions of patients worldwide every year. Aquaporin drug discovery has made little progress, possibly due to a range of assumptions including the intrinsic non-druggability of the aquaporin pore, compounded by issues with the reproducibility of current assays. We challenge the persistent idea that aquaporins are not druggable; the field is still in its infancy and much progress is yet to be made. Viable routes to inhibition of aquaporin function have recently been identified, including targeting their regulation as well as their pores. Identifying new aquaporin-targeted drugs for conditions associated with disrupted water and solute homeostasis will meet an urgent, unmet clinical need as no pharmacological interventions are currently available. Inhibition of hydrogen peroxide permeation through AQP1 provides a new approach to treating hypertrophic cardiomyopathies. Inhibition of AQP4 localization with the licensed drug trifluoperazine provides compelling evidence for a new approach to treating CNS edema.

To study the function and structure of membrane proteins, high quantities of pure and stable protein are needed. One of the first hurdles in accomplishing this is expression of the membrane protein at high levels and in a functional state. Membrane proteins are naturally expressed at low levels, so finding a suitable host for overexpression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein that is able to transport a range of organic anionic compounds (both endogenous and xenobiotic) out of the cell. This versatile transporter has been linked with extracellular signaling pathways and cellular protection, along with conferring drug resistance in cancers. Here we report the use of MRP4 as a case study to be expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Interestingly, using the baculovirus expression system with Sf9 insect cells produced the highest protein yields. Vesicular transport assays were used to confirm that MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabeled substrates. MRP4 transported fluo-cAMP in an ATP-dependent manner. The specificity of functional expression of MRP4 was validated by the use of nonhydrolyzable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilization and purification in order to better understand its function and structure.

Aquaporins (AQPs) are a ubiquitous family of transmembrane water channel proteins. A subgroup of AQP water channels also facilitates transmembrane diffusion of small, polar solutes. A constriction within the pore, the aromatic/arginine (ar/R) selectivity filter, is thought to control solute permeability: previous studies on single representative water channel proteins suggest narrow channels conduct water, whilst wider channels permit passage of solutes. To assess this model of selectivity, we used mutagenesis, permeability measurements and in silico comparisons of water-specific as well as glycerol-permeable human AQPs. Our studies show that single amino acid substitutions in the selectivity filters of AQP1, AQP4 and AQP3 differentially affect glycerol and urea permeability in an AQP-specific manner. Comparison between in silico-calculated channel cross-sectional areas and in vitro permeability measurements suggests that selectivity filter cross-sectional area predicts urea but not glycerol permeability. Our data show that substrate discrimination in water channels depends on a complex interplay between the solute, pore size, and polarity, and that using single water channel proteins as representative models has led to an underestimation of this complexity.

The environmentally-friendly, economically-viable production of ethanol from cellulosic biomass remains a major contemporary challenge. Much work has been done on the disruption of cellulosic biomass structure, the production of enzymes for the conversion of cellulose and hemicellulose into simple sugars that can be fermented by bacteria or yeast, and the metabolic engineering of ethanol-producing microbes. The results of these studies have enabled the transition from laboratory to industrial scale of cellulosic ethanol production. Notably, however, current processes use free microbial cells in batch reactors. This review highlights the advantages of using immobilized and co-immobilized cells together with continuous bioreactor configurations. These developments have the potential to improve both the yield and the green credentials of cellulosic ethanol production in modern industrial settings.

Membrane proteins (MPs) are important drug discovery targets for a wide range of diseases. However, elucidating the structure and function of native MP is notoriously challenging as their original structure has to be maintained once removed from the lipid bilayer. Conventionally, detergents have been used to solubilize MP with varying degrees of success concerning MP stability. To try to address this, new, more stabilizing agents have been developed, such as calixarene-based detergents and styrene–maleic acid (SMA) copolymer. Calixarene-based detergents exhibit enhanced solubilizing and stabilizing properties compared with conventional detergents, whereas SMA is able to extract MPs with their surrounding lipids, forming a nanodisc structure. Here we report a comparative study using classical detergents, calixarene-based detergents, and SMA to assess the solubilization and stabilization of the human ABC transporter MRP4 (multidrug resistance protein 4/ABCC4). We show that both SMA and calixarene-based detergents have a higher solubility efficiency (at least 80%) than conventional detergents, and show striking overstabilization features of MRP4 (up to 70 °C) with at least 30 °C stability improvement in comparison with the best conventional detergents. These solubilizing agents were successfully used to purify aggregate-free, homogenous and stable MRP4, with sevenfold higher yield for C4C7 calixarene detergent in comparison with SMA. This work paves the way to MRP4 structural and functional investigations and illustrates once more the high value of using calixarene-based detergent or SMA as versatile and efficient tools to study MP, and eventually enable drug discovery of challenging and highly druggable targets.

Aquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4 that occurs in response to osmotically-driven changes in cell swelling in the central nervous system. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca2+-dependent CaM-binding properties of synthetic and recombinant AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1H, 15N-HSQC NMR with 15N-labeled CaM. The N-terminal CBD peptide of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 μM. CaM bound two C-terminal AQP4 peptides with interactions observed for both the C- and N-lobes of CaM (Kd1: 3.6 μM, Kd2: 113.6 μM, respectively). A recombinant AQP4 protein domain (rAQP4ct, containing the entire cytosolic C-terminal domain sequence) bound CaM in a 1:1 binding mode with a Kd of 6.1 μM. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data suggest that this unique adapter protein binding mode of CaM and AQP4 may be an important regulatory mechanism for the vesicular trafficking of AQP4.

Neurodegenerative diseases (NDs) including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease are incurable and affect millions of people worldwide. The development of treatments for this unmet clinical need is a major global research challenge. Computer-aided drug design (CADD) methods minimize the huge number of ligands that could be screened in biological assays, reducing the cost, time, and effort required to develop new drugs. In this review, we provide an introduction to CADD and examine the progress in applying CADD and other molecular docking studies to NDs. We provide an updated overview of potential therapeutic targets for various NDs and discuss some of the advantages and disadvantages of these tools.

Aquaporin water channels facilitate the bi-directional flow of water and small, neutral solutes down an osmotic gradient in all kingdoms of life. Over the last two decades, the availability of high-quality protein has underpinned progress in the structural and functional characterization of these water channels. In particular, recombinant protein technology has guaranteed the supply of aquaporin samples that were of sufficient quality and quantity for further study. Here we review the features of successful expression, purification and characterization strategies that have underpinned these successes and that will drive further breakthroughs in the field. Overall, Escherichia coli is a suitable host for prokaryotic isoforms, while Pichia pastoris is the most commonly-used recombinant host for eukaryotic variants. Generally, a two-step purification procedure is suitable after solubilization in glucopyranosides and most structures are determined by X-ray following crystallization.

In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein–lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein–protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.

Tetraspanins exert a wide range of cellular functions of broad medical importance. Despite this, their biophysical characteristics are incompletely understood. Only two high-resolution structures of full-length tetraspanins have been solved. One is that of human CD81, which is involved in the infectivity of human pathogens including influenza, HIV, the malarial Plasmodium parasite and hepatitis C virus (HCV). The CD81 crystal structure identifies a cholesterol-binding pocket, which has been suggested to be important in the regulation of tetraspanin function. Here we investigate the use of styrene-maleic anhydride co-polymers (SMA) for the solubilisation and purification of CD81 within a lipid environment. When CD81 was expressed in the yeast Pichia pastoris, it could be solubilised and purified using SMA2000. This SMALP-encapsulated CD81 retained its native folded structure, as determined by the binding of two conformation-sensitive anti-CD81 antibodies. Analysis by size exclusion chromatography revealed two distinct populations of CD81, only one of which bound the HCV glycoprotein, E2. Optimization of expression and buffer conditions increased the proportion of E2-binding competent CD81 protein. Mass spectrometry analysis indicated that the lipid environment surrounding CD81 is enriched with negatively charged lipids. These results establish a platform to study the influence of protein-lipid interactions in tetraspanin biology., Graphical abstract Unlabelled Image, Highlights • CD81 expressed in Pichia pastoris can be solubilised and purified using SMA polymer. • SMALP-encapsulated CD81 retains native folded structure. • Expression and buffer conditions can be optimized to improve protein quality. • The lipid environment surrounding CD81 is enriched with negatively charged lipids.

ABCC1 and ABCC4 utilize energy from ATP hydrolysis to transport many different molecules, including drugs, out of the cell and, as such, have been implicated in causing drug resistance. However recently, because of their ability to transport signaling molecules and inflammatory mediators, it has been proposed that ABCC1 and ABCC4 may play a role in the hallmarks of cancer development and progression, independent of their drug efflux capabilities. Breast cancer is the most common cancer affecting women. In this study, the aim was to investigate whether ABCC1 or ABCC4 play a role in the proliferation or migration of breast cancer cell lines MCF-7 (luminal-type, receptor-positive) and MDA-MB-231 (basal-type, triple-negative). The effects of small molecule inhibitors or siRNA-mediated knockdown of ABCC1 or ABCCC4 were measured. Colony formation assays were used to assess the clonogenic capacity, MTT assays to measure the proliferation, and scratch assays and Transwell assays to monitor the cellular migration. The results showed a role for ABCC1 in cellular proliferation, whilst ABCC4 appeared to be more important for cellular migration. ELISA studies implicated cAMP and/or sphingosine-1-phosphate efflux in the mechanism by which these transporters mediate their effects. However, this needs to be investigated further, as it is key to understand the mechanisms before they can be considered as targets for treatment.

Aquaporins and aquaglyceroporins are membrane channels that allow the permeation of water and certain other small solutes across the cell membrane, or in the case of AQP6, AQP11 and AQP12A, intracellular membranes, such as vesicles and the endoplasmic reticulum membrane [17]. Since the isolation and cloning of the first aquaporin (AQP1) [21], 12 additional mammalian members of the family have been identified, although little is known about the functional properties of one of these (AQP12A; Q8IXF9) and it is thus not tabulated. The other 12 aquaporins can be broadly divided into three families: orthodox aquaporins (AQP0,-1,-2,-4,-5, -6 and -8) permeable mainly to water, but for some additional solutes [5]; aquaglyceroporins (AQP3,-7 -9 and -10), additionally permeable to glycerol and for some isoforms urea [16], and superaquaporins (AQP11 and 12) located within cells [14]. Some aquaporins also conduct ammonia and/or H2O2 giving rise to the terms 'ammoniaporins' ('aquaammoniaporins') and 'peroxiporins', respectively. Aquaporins are impermeable to protons and other inorganic and organic cations, with the possible exception of AQP1 [16]. One or more members of this family of proteins have been found to be expressed in almost all tissues of the body [reviewed in Yang (2017) [27]]. AQPs are involved in numerous processes that include systemic water homeostasis, adipocyte metabolism, brain oedema, cell migration and fluid secretion by epithelia and loss of function mutations of some human AQPs, or their disruption by autoantibodies further underscore their importance [reviewed by Verkman et al. (2014) [24], Kitchen et al. (2105) [16]]. Functional AQPs exist as homotetramers that are the water conducting units wherein individual AQP subunits (each a protomer) have six transmembrane helices and two half helices that constitute a seventh 'pseudotransmembrane domain' that surrounds a narrow water conducting channel [17]. In addition to the four pores contributed by the protomers, an additional hydrophobic pore exists within the center of the complex [17] that may mediate the transport of gases (e.g. O2, CO2, NO) and cations (the central pore is the proposed transport pathway for cations through AQP1) by some AQPs [8, 15]. Although numerous small molecule inhibitors of aquaporins, particularly APQ1, have been reported primarily from Xenopus oocyte swelling assays, the activity of most has subsequently been disputed upon retesting using assays of water transport that are less prone to various artifacts [6] and they are therefore excluded from the tables [see Tradtrantip et al. (2017) [23] for a review].

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques., Highlights • 1713% increase in unique membrane protein structures since 2000. • Variety of expression methods available to GPCR researchers, now including the eyes of Drosophila melanogaster. • Development of novel solubilisation strategies including the styrene maleic acid co-polymer.

Summary Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies., Graphical Abstract, Highlights • Hypoxia triggers an increase in AQP4-mediated flux of water into astrocytes • Translocation of AQP4 to the astrocyte cell surface drives increased water flux • AQP4 cell-surface localization is mediated by a CaM- and PKA-dependent mechanism • Inhibition of AQP4 localization with the licensed drug TFP halts CNS edema in rats, Modulating the subcellular localization of the water channel protein AQP4 may be a therapeutic option for treatment of brain and spinal cord edemas.

Recent research in the aquaporin (AQP) field has identified a role for diverse AQPs in extracellular vesicles (EV). Though still in its infancy, there is a growing body of knowledge in the area; AQPs in EV have been suggested as biomarkers for disease, as drug targets and show potential as therapeutics. To advance further in this field, AQPs in EV must be better understood. Here we summarize current knowledge of the presence and function of AQPs in EV and hypothesise their roles in health and disease.

Aquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca2+-dependent CaM-binding properties of AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1H, 15N-HSQC NMR with 15N-labeled CaM. The N-terminal CBD of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 μM. The C-terminal AQP4 peptide interacted with both the C- and N-lobes of CaM (2:1 binding ratio; Kd1: 3.6 μM, Kd2: 113.6 μM, respectively). A recombinant AQP4 protein domain (recAQP4CT, containing the entire cytosolic C-terminal sequence) bound CaM in a 1:1 binding mode with a Kd of 6.1 μM. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data support a unique adapter protein binding mode for CaM with AQP4.

Human aquaporin 4 (AQP4) is the primary water channel protein in brain astrocytes. Hypothermia is known to cause astrocyte swelling in culture, but the precise role of AQP4 in this process is unknown. Primary human cortical astrocytes were cultured under hypothermic (32 °C) or normothermic (37 °C) conditions. AQP4 transcript, total protein and surface‐localized protein were quantified using RT‐qPCR, sandwich ELISA with whole cell lysates or cell surface biotinylation, followed by ELISA analysis of the surface‐localized protein, respectively. Four‐hour mild hypothermic treatment increased the surface localization of AQP4 in human astrocytes to 155 ± 4% of normothermic controls, despite no change in total protein expression levels. The hypothermia‐mediated increase in AQP4 surface abundance on human astrocytes was blocked using either calmodulin antagonist (trifluoperazine, TFP); TRPV4 antagonist, HC‐067047 or calcium chelation using EGTA‐AM. The TRPV4 agonist (GSK1016790A) mimicked the effect of hypothermia compared with untreated normothermic astrocytes. Hypothermia led to an increase in surface localization of AQP4 in human astrocytes through a mechanism likely dependent on the TRPV4 calcium channel and calmodulin activation. Understanding the effects of hypothermia on astrocytic AQP4 cell surface expression may help develop new treatments for brain swelling based on an in‐depth mechanistic understanding of AQP4 translocation.