The C-terminal activating domain promotes Panx1 channel opening.

Pannexin 1 (Panx1) constitutes a large pore channel responsible for the release of ATP from apoptotic cells. Strong evidence indicates that caspase-mediated cleavage of the C-terminus promotes the opening of the Panx1 channel by unplugging the pore. However, this simple pore-plugging mechanism alone cannot account for the observation that a Panx1 construct ending before the caspase cleavage site remains closed. Here, we show that a helical region located immediately before the caspase cleavage site, referred to as the "C-terminal activating domain (CAD)," plays a pivotal role in facilitating Panx1 activation. Electrophysiology and mutagenesis studies uncovered that two conserved leucine residues within the CAD plays a pivotal role. Cryo-EM analysis of the construct ending before reaching the CAD demonstrated that the N-terminus extends into an intracellular pocket. In contrast, the construct including the CAD revealed that this domain occupies the intracellular pocket, causing the N-terminus to flip upward within the pore. Analysis of electrostatic free energy landscape in the closed conformation indicated that the intracellular side of the ion permeation pore may be occupied by anions like ATP, creating an electrostatic barrier for anions attempting to permeate the pore. When the N-terminus flips up, it diminishes the positively charged surface, thereby reducing the drive to accumulate anions inside the pore. This dynamic change in the electrostatic landscape likely contributes to the selection of permeant ions. Collectively, these experiments put forth a novel mechanism in which C-terminal cleavage liberates the CAD, causing the repositioning of the N-terminus to promote Panx1 channel opening.

ATP-release pannexin channels are gated by lysophospholipids.

Adenosine triphosphate (ATP) serves as an extracellular messenger that mediates diverse cell-to-cell communication. Compelling evidence supports that ATP is released from cells through pannexins, a family of heptameric large pore-forming channels. However, the activation mechanisms that trigger ATP release by pannexins remain poorly understood. Here, we discover lysophospholipids as endogenous pannexin activators, using activity-guided fractionation of mouse tissue extracts combined with untargeted metabolomics and electrophysiology. We show that lysophospholipids directly and reversibly activate pannexins in the absence of other proteins. Molecular docking, mutagenesis, and single-particle cryo-EM reconstructions suggest that lysophospholipids open pannexin channels by altering the conformation of the N-terminal domain. Our results provide a connection between lipid metabolism and ATP signaling, both of which play major roles in inflammation and neurotransmission. One-Sentence Summary: Untargeted metabolomics discovers a class of messenger lipids as endogenous activators of membrane channels important for inflammation and neurotransmission.

GCP16 stabilizes the DHHC9 subfamily of protein acyltransferases through a conserved C-terminal cysteine motif.

Protein S-acylation is a reversible lipid post-translational modification that allows dynamic regulation of processes such as protein stability, membrane association, and localization. Palmitoyltransferase ZDHHC9 (DHHC9) is one of the 23 human DHHC acyltransferases that catalyze protein S-acylation. Dysregulation of DHHC9 is associated with X-linked intellectual disability and increased epilepsy risk. Interestingly, activation of DHHC9 requires an accessory protein-GCP16. However, the exact role of GCP16 and the prevalence of a requirement for accessory proteins among other DHHC proteins remain unclear. Here, we report that one role of GCP16 is to stabilize DHHC9 by preventing its aggregation through formation of a protein complex. Using a combination of size-exclusion chromatography and palmitoyl acyltransferase assays, we demonstrate that only properly folded DHHC9-GCP16 complex is enzymatically active in vitro. Additionally, the ZDHHC9 mutations linked to X-linked intellectual disability result in reduced protein stability and DHHC9-GCP16 complex formation. Notably, we discovered that the C-terminal cysteine motif (CCM) that is conserved among the DHHC9 subfamily (DHHC14, -18, -5, and -8) is required for DHHC9 and GCP16 complex formation and activity in vitro. Co-expression of GCP16 with DHHCs containing the CCM improves DHHC protein stability. Like DHHC9, DHHC14 and DHHC18 require GCP16 for their enzymatic activity. Furthermore, GOLGA7B, an accessory protein with 75% sequence identity to GCP16, improves protein stability of DHHC5 and DHHC8, but not the other members of the DHHC9 subfamily, suggesting selectivity in accessory protein interactions. Our study supports a broader role for GCP16 and GOLGA7B in the function of human DHHCs.

Methods for Studying Cholesterol-Dependent Regulation of P2X7 Receptors.

Cholesterol dynamically regulates P2X7 receptor function in both physiological and pathological conditions. Studies suggest that cholesterol suppresses P2X7 receptor activity through direct binding or through indirect effects on the biophysical properties of the membrane. Notably, the palmitoylated C-terminus seems to counteract the action of cholesterol to make it less inhibitory. However, the mechanism underlying cholesterol-dependent regulation of P2X7 receptor remains unclear. Here we describe detailed methods that facilitate the quantification of P2X7 channel activity while controlling the amount of cholesterol in the system. We will first describe the use of methyl-ß-cyclodextrin (MCD), a cyclic oligosaccharide consisting of seven glucose monomers, to decrease or increase plasma membrane cholesterol levels. We will then describe protocols for the reconstitution of purified P2X7 in proteoliposomes of defined lipid composition. These methods can be combined with commonly used techniques such as dye-uptake assays or electrophysiology. We also describe a fluorescence assay to measure cholesterol-binding to P2X7. These approaches are complementary to cryo-EM or molecular dynamics simulations, which are also powerful tools for investigating cholesterol-P2X7 interactions. An improved understanding of the mechanisms of action of cholesterol on P2X7 may contribute to elucidate the roles of this receptor in ageing, inflammation, and cancer, whose progression correlates with the level of cholesterol.

On the molecular nature of large-pore channels.

Membrane transport is a fundamental means to control basic cellular processes such as apoptosis, inflammation, and neurodegeneration and is mediated by a number of transporters, pumps, and channels. Accumulating evidence over the last half century has shown that a type of so-called "large-pore channel" exists in various tissues and organs in gap-junctional and non-gap-junctional forms in order to flow not only ions but also metabolites such as ATP. They are formed by a number of protein families with little or no evolutionary linkages including connexin, innexin, pannexin, leucine-rich repeat-containing 8 (LRRC8), and calcium homeostasis modulator (CALHM). This review summarizes the history and concept of large-pore channels starting from connexin gap junction channels to the more recent developments in innexin, pannexin, LRRC8, and CALHM. We describe structural and functional features of large-pore channels that are crucial for their diverse functions on the basis of available structures.

The Cryo-EM structure of pannexin 1 reveals unique motifs for ion selection and inhibition.

Pannexins are large-pore forming channels responsible for ATP release under a variety of physiological and pathological conditions. Although predicted to share similar membrane topology with other large-pore forming proteins such as connexins, innexins, and LRRC8, pannexins have minimal sequence similarity to these protein families. Here, we present the cryo-EM structure of a frog pannexin 1 (Panx1) channel at 3.0 Å. We find that Panx1 protomers harbor four transmembrane helices similar in arrangement to other large-pore forming proteins but assemble as a heptameric channel with a unique constriction formed by Trp74 in the first extracellular loop. Mutating Trp74 or the nearby Arg75 disrupt ion selectivity, whereas altering residues in the hydrophobic groove formed by the two extracellular loops abrogates channel inhibition by carbenoxolone. Our structural and functional study establishes the extracellular loops as important structural motifs for ion selectivity and channel inhibition in Panx1.

CAKUT and Autonomic Dysfunction Caused by Acetylcholine Receptor Mutations.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life, and in utero obstruction to urine flow is a frequent cause of secondary upper urinary tract malformations. Here, using whole-exome sequencing, we identified three different biallelic mutations in CHRNA3, which encodes the α3 subunit of the nicotinic acetylcholine receptor, in five affected individuals from three unrelated families with functional lower urinary tract obstruction and secondary CAKUT. Four individuals from two families have additional dysautonomic features, including impaired pupillary light reflexes. Functional studies in vitro demonstrated that the mutant nicotinic acetylcholine receptors were unable to generate current following stimulation with acetylcholine. Moreover, the truncating mutations p.Thr337Asnfs∗81 and p.Ser340∗ led to impaired plasma membrane localization of CHRNA3. Although the importance of acetylcholine signaling in normal bladder function has been recognized, we demonstrate for the first time that mutations in CHRNA3 can cause bladder dysfunction, urinary tract malformations, and dysautonomia. These data point to a pathophysiologic sequence by which monogenic mutations in genes that regulate bladder innervation may secondarily cause CAKUT.

The weak voltage dependence of pannexin 1 channels can be tuned by N-terminal modifications.

Pannexins are a family of ATP release channels important for physiological and pathological processes like blood pressure regulation, epilepsy, and neuropathic pain. To study these important channels in vitro, voltage stimulation is the most common and convenient tool, particularly for pannexin 1 (Panx1). However, whether Panx1 is a voltage-gated channel remains controversial. Here, we carefully examine the effect of N-terminal modification on voltage-dependent Panx1 channel activity. Using a whole-cell patch-clamp recording technique, we demonstrate that both human and mouse Panx1, with their nativeN termini, give rise to voltage-dependent currents, but only at membrane potentials larger than +100 mV. This weak voltage-dependent channel activity profoundly increases when a glycine-serine (GS) motif is inserted immediately after the first methionine. Single-channel recordings reveal that the addition of GS increases the channel open probability as well as the number of unitary conductance classes. We also find that insertions of other amino acid(s) at the same position mimics the effect of GS. On the other hand, tagging the N terminus with GFP abolishes voltage-dependent channel activity. Our results suggest that Panx1 is a channel with weak voltage dependence whose activity can be tuned by N-terminal modifications.

Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells.

The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is then purified in a buffer containing dodecyl maltoside using Strep-Tactin affinity chromatography. Following enzymatic cleavage of the attached GFP and Strep-tag by thrombin, the P2X7 receptor is isolated using size exclusion chromatography. This method typically yields ~2 mg of purified protein from 6 L of Sf9 culture. Purified protein can be stored with a buffer containing 15% glycerol at 4 °C for at least 2 months and used for a variety of functional and structural studies (Karasawa and Kawate, 2016).

The P2X7 receptor forms a dye-permeable pore independent of its intracellular domain but dependent on membrane lipid composition.

The P2X7 receptor mediates extracellular ATP signaling implicated in the development of devastating diseases such as chronic pain and cancer. Activation of the P2X7 receptor leads to opening of the characteristic dye-permeable membrane pore for molecules up to ~900 Da. However, it remains controversial what constitutes this peculiar pore and how it opens. Here we show that the panda receptor, when purified and reconstituted into liposomes, forms an intrinsic dye-permeable pore in the absence of other cellular components. Unexpectedly, we found that this pore opens independent of its unique C-terminal domain. We also found that P2X7 channel activity is facilitated by phosphatidylglycerol and sphingomyelin, but dominantly inhibited by cholesterol through direct interactions with the transmembrane domain. In combination with cell-based functional studies, our data suggest that the P2X7 receptor itself constitutes a lipid-composition dependent dye-permeable pore, whose opening is facilitated by palmitoylated cysteines near the pore-lining helix.

P2X Receptor Activation.

Extracellular ATP-gated P2X receptors are trimeric non-selective cation channels important for many physiological events including immune response and neural transmission. These receptors belong to a unique class of ligand-gated ion channels composed of only six transmembrane helices and a relatively small extracellular domain that harbors three ATP-binding pockets. The crystal structures of P2X receptors, including the recent P2X3 structures representing three different stages of the gating cycle, have provided a compelling structural foundation for understanding how this class of ligand-gated ion channels function. These structures, in combination with numerous functional studies ranging from classic mutagenesis and electrophysiology to modern optogenetic pharmacology, have uncovered unique molecular mechanisms of P2X receptor function. This review article summarizes the current knowledge in P2X receptor activation, especially focusing on the mechanisms underlying ATP-binding, conformational changes in the extracellular domain, and channel gating and desensitization.

Structural basis for subtype-specific inhibition of the P2X7 receptor.

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions on protein activity and the roles of membrane proteins in disease pathways.

Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop.

Pannexin1 (Panx1) is an ATP release channel important for controlling immune responses and synaptic strength. Various stimuli including C-terminal cleavage, a high concentration of extracellular potassium, and voltage have been demonstrated to activate Panx1. However, it remains unclear how Panx1 senses and integrates such diverse stimuli to form an open channel. To provide a clue on the mechanism underlying Panx1 channel gating, we investigated the action mechanism of carbenoxolone (CBX), the most commonly used small molecule for attenuating Panx1 function triggered by a wide range of stimuli. Using a chimeric approach, we discovered that CBX reverses its action polarity and potentiates the voltage-gated channel activity of Panx1 when W74 in the first extracellular loop is mutated to a nonaromatic residue. A systematic mutagenesis study revealed that conserved residues in this loop also play important roles in CBX function, potentially by mediating CBX binding. We extended our experiments to other Panx1 inhibitors such as probenecid and ATP, which also potentiate the voltage-gated channel activity of a Panx1 mutant at position 74. Notably, probenecid alone can activate this mutant at a resting membrane potential. These data suggest that CBX and other inhibitors, including probenecid, attenuate Panx1 channel activity through modulation of the first extracellular loop. Our experiments are the first step toward identifying a previously unknown mode of CBX action, which provide insight into the role of the first extracellular loop in Panx1 channel gating.

Ion access pathway to the transmembrane pore in P2X receptor channels.

P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

Pore-opening mechanism in trimeric P2X receptor channels.

The opening of ion channels in response to ligand binding, voltage or membrane stretch underlies electrical and chemical signalling throughout biology. Two structural classes of pore-opening mechanisms have been established, including bending of pore-lining helices in the case of tetrameric cation channels, or tilting of such helices in mechanosensitive channels. In this paper, we explore how the structure of the pore changes during opening in P2X receptors by measuring the modification of introduced cysteine residues in transmembrane helices by thiol-reactive reagents, and by engineering metal bridges. Our results are consistent with the X-ray structure of the closed state, and demonstrate that expansion of the gate region in the external pore is accompanied by a significant narrowing of the inner pore, indicating that pore-forming helices straighten on ATP binding to open the channel. This unique pore-opening mechanism has fundamental implications for the role of subunit interfaces in the gating mechanism of P2X receptors and points to a role of the internal pore in ion permeation.

Crystal structure of the ATP-gated P2X(4) ion channel in the closed state.

P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X(4) receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit-subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in beta-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the 'gate' is defined by an approximately 8 A slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening.

Pore architecture and ion sites in acid-sensing ion channels and P2X receptors.

Acid-sensing ion channels are proton-activated, sodium-selective channels composed of three subunits, and are members of the superfamily of epithelial sodium channels, mechanosensitive and FMRF-amide peptide-gated ion channels. These ubiquitous eukaryotic ion channels have essential roles in biological activities as diverse as sodium homeostasis, taste and pain. Despite their crucial roles in biology and their unusual trimeric subunit stoichiometry, there is little knowledge of the structural and chemical principles underlying their ion channel architecture and ion-binding sites. Here we present the structure of a functional acid-sensing ion channel in a desensitized state at 3 A resolution, the location and composition of the approximately 8 A 'thick' desensitization gate, and the trigonal antiprism coordination of caesium ions bound in the extracellular vestibule. Comparison of the acid-sensing ion channel structure with the ATP-gated P2X(4) receptor reveals similarity in pore architecture and aqueous vestibules, suggesting that there are unanticipated yet common structural and mechanistic principles.

Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins.

Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.

Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters.

Na+/Cl--dependent transporters terminate synaptic transmission by using electrochemical gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compounds, and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homologue of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1-5 related to 6-10 by a pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helices, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.