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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.

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.

N-Methyl-D-aspartate receptors (NMDARs) play critical roles in the central nervous system. Their heterotetrameric composition generates subtypes with distinct functional properties and spatio-temporal distribution in the brain, raising the possibility for subtype-specific targeting by pharmacological means for treatment of neurological diseases. While specific compounds for GluN2A and GluN2B-containing NMDARs are well established, those that target GluN2C and GluN2D are currently underdeveloped with low potency and uncharacterized binding modes. Here, using electrophysiology and X-ray crystallography, we show that UBP791 ((2S*,3R*)-1-(7-(2-carboxyethyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) inhibits GluN2C/2D with 40-fold selectivity over GluN2A-containing receptors, and that a methionine and a lysine residue in the ligand binding pocket (GluN2D-Met763/Lys766, GluN2C-Met736/Lys739) are the critical molecular elements for the subtype-specific binding. These findings led to development of UBP1700 ((2S*,3R*)-1-(7-(2-carboxyvinyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) which shows over 50-fold GluN2C/2D-selectivity over GluN2A with potencies in the low nanomolar range. Our study shows that the l-glutamate binding site can be targeted for GluN2C/2D-specific inhibition., Selectively inhibiting N-Methyl-D-aspartate receptors (NMDARs) containing the GluN2C/2D subunits has been challenging. Here, using electrophysiology and X-ray crystallography, authors show that compounds UBP791 and UBP1700 show over 40- and 50-fold selectivity for GluN2C/2D compared to GluN2A.

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 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 gate formed by Trp74 in the 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 the unique structural determinants for channel gating and inhibition in Panx1 thereby providing the founding model to study pannexins.

Summary Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation., Highlights • The MDGA1 extracellular region has an unusual triangular multi-domain arrangement • The NL1-MDGA1 complex structure reveals how MDGA proteins block neurexin binding • MDGA1 and MDGA2 bind all NL isoforms, a process fine-tuned by alternative splicing • MDGA1 and MDGA2 suppress NL synaptogenic activity in a concentration-dependent manner, Elegheert et al. present the crystal structure of the autism-linked post-synaptic protein MDGA in complex with the synapse organizer neuroligin, providing a structural and mechanistic basis for potentially brain-wide modulation of synaptic neuroligin-neurexin signaling by MDGA proteins.

Competitive antagonists against N-methyl-D-aspartate (NMDA) receptors have played critical roles throughout the history of neuropharmacology and basic neuroscience. There are currently numerous NMDA receptor antagonists containing a variety of chemical groups. Among those compounds, a GluN2-specific antagonist, (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl-phosphonic acid (NVP-AAM077), contains a unique combination of a dioxoquinoxalinyl ring, a bromophenyl group, and a phosphono group. In this study, we present the crystal structure of the isolated ligand-binding domain of the GluN1-GluN2A NMDA receptor in complex with the GluN1 agonist glycine and the GluN2A antagonist NVP-AAM077. The structure shows placement of the dioxoquinoxalinyl ring and the phosphono group of NVP-AAM077 in the glutamate-binding pocket in GluN2A and the novel interaction between the bromophenyl group and GluN1-Glu781 at the GluN1-GluN2A subunit interface. Site-directed mutagenesis of GluN1-Glu781 reduced the potency of inhibition by NVP-AAM077, thus confirming the involvement of the GluN1 subunit for binding of NVP-AAM077. The unique antagonist-binding pattern shown in this study provides a novel dimension to design and create antagonists with potential therapeutic values.

N-methyl-d-aspartate (NMDA) receptors are ligand-gated ion channels assembled from GluN1 and GluN2 subunits. We used a series of N-hydroxypyrazole-5-glycine (NHP5G) partial agonists at the GluN2 glutamate binding site as tools to study activation of GluN1/GluN2A and GluN1/GluN2D NMDA receptor subtypes. Using two-electrode voltage-clamp electrophysiology, fast-application patch-clamp, and single-channel recordings, we show that propyl- and ethyl-substituted NHP5G agonists have a broad range of agonist efficacies relative to the full agonist glutamate (

NMDA receptors are tetrameric complexes of GluN1, GluN2A-D, and GluN3A-B subunits and are involved in normal brain function and neurologic disorders. We identified a novel class of stereoselective pyrrolidinone (PYD) positive allosteric modulators for GluN2C-containing NMDA receptors, exemplified by methyl 4-(3-acetyl-4-hydroxy-1-[2-(2-methyl-1H-indol-3-yl)ethyl]-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)benzoate. Here we explore the site and mechanism of action of a prototypical analog, PYD-106, which at 30 μM does not alter responses of NMDA receptors containing GluN2A, GluN2B, and GluN2D and has no effect on AMPA [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid] and kainate receptors. Coapplication of 50 μM PYD-106 with a maximally effective concentration of glutamate and glycine increases the response of GluN1/GluN2C NMDA receptors in HEK-293 cells to 221% of that obtained in the absence of PYD (taken as 100%). Evaluation of the concentration dependence of this enhancement revealed an EC50 value for PYD of 13 μM. PYD-106 increased opening frequency and open time of single channel currents activated by maximally effective concentrations of agonist but only had modest effects on glutamate and glycine EC50. PYD-106 selectively enhanced the responses of diheteromeric GluN1/GluN2C receptors but not triheteromeric GluN1/GluN2A/GluN2C receptors. Inclusion of residues encoded by GluN1-exon 5 attenuated the effects of PYD. Three GluN2C residues (Arg194, Ser470, Lys470), at which mutagenesis virtually eliminated PYD function, line a cavity at the interface of the ligand binding and the amino terminal domains in a homology model of GluN1/GluN2C built from crystallographic data on GluN1/GluN2B. We propose that this domain interface constitutes a new allosteric modulatory site on the NMDA receptor.

Background: Although tacrolimus-based immunosuppression has made intestinal transplantation feasible, the risk of the requisite chronic high-dose treatment has inhibited the widespread use of these procedures. We have examined our 1990–1997 experience to determine whether immunomodulatory strategies to improve outlook could be added to drug treatment. Study Design: Ninety-eight consecutive patients (59 children, 39 adults) with a panoply of indications received 104 allografts under tacrolimus-based immunosuppression: intestine only (n = 37); liver and intestine (n = 50); or multivisceral (n = 17). Of the last 42 patients, 20 received unmodified adjunct donor bone marrow cells; the other 22 were contemporaneous control patients. Results: With a mean followup of 32 ± 26 months (range, 1–86 months), 12 recipients (3 intestine only, 9 composite grafts) are alive with good nutrition beyond the 5-year milestone. Forty-seven (48%) of the total group survive bearing grafts that provide full (91%) or partial (9%) nutrition. Actuarial patient survival at 1 and 5 years (72% and 48%, respectively) was similar with isolated intestinal and composite graft recipients, but the loss rate of grafts from rejection was highest with intestine alone. The best results were in patients between 2 and 18 years of age (68% at 5 years). Adjunct bone marrow did not significantly affect the incidence of graft rejection, B-cell lymphoma, or the rate or severity of graft-versus-host disease. Conclusions: These results demonstrate that longterm rehabilitation similar to that with the other kinds of organ allografts is achievable with all three kinds of intestinal transplant procedures, that the morbidity and mortality is still too high for their widespread application, and that the liver is significantly but marginally protective of concomitantly engrafted intestine. Although none of the endpoints were markedly altered by donor leukocyte augmentation (and chimerism) with bone marrow, establishment of the safety of this adjunct procedure opens the way to further immune modulation strategies that can be added to the augmentation protocol.

Most fast excitatory synaptic transmission in the nervous system is mediated by glutamate acting through ionotropic glutamate receptors (iGluRs). iGluRs (AMPA, kainate, and NMDA receptor subtypes) are tetrameric assemblies, formed as a dimer of dimers. Still, the mechanism underlying tetramerization--the necessary step for the formation of functional receptors that can be inserted into the plasma membrane--is unknown. All eukaryotic compared to prokaryotic iGluR subunits have an additional transmembrane segment, the M4 segment, which positions the physiologically critical C-terminal domain on the cytoplasmic side of the membrane. AMPA receptor (AMPAR) subunits lacking M4 do not express on the plasma membrane. Here, we show that these constructs are retained in the endoplasmic reticulum, the major cellular compartment mediating protein oligomerization. Using approaches to assay the native oligomeric state of AMPAR subunits, we find that subunits lacking M4 or containing single amino acid substitutions along an "interacting" face of the M4 helix that block surface expression no longer tetramerize in either homomeric or heteromeric assemblies. In contrast, subunit dimerization appears to be largely intact. These experiments define the M4 segment as a unique functional unit in AMPARs that is required for the critical dimer-to-tetramer transition.

Between April 1993 and May 1995, 17 adult orthotopic liver transplant recipients were found to have early hepatic artery thrombosis (HAT) after a median of 7 postoperative days (mean, 11). The HAT was diagnosed in all cases by duplex ultrasound. Thrombectomy was performed with urgent revascularization (UR), using an interposition arterial graft procured from the cadaveric liver donor, and arterial patency was verified with intraoperative angiography. In seven cases, intra-arterial urokinase was administered after the thrombectomy. Fifteen (88%) of the livers remained arterialized throughout the follow-up period (median, 15 months); the remaining two patients developed recurrent HAT after 6 and 8 months. Although there was a high rate of subsequent complications, 11 (65%) of the patients are alive without retransplantation, with a mean follow-up of 17 months. Despite having a patent hepatic artery, the remaining six patients (35%) died from infectious complications that usually were present before the UR. Thus, UR effectively restored arterial inflow in 88% of the patients with early HAT. The ultimate outcome was determined mainly by the presence of intra-abdominal complications at the time of UR. In conclusion, UR, rather than retransplantation, should be considered the prime treatment option for patients who develop early posttransplant HAT.

Background The purpose of the study was to quantify changes in the quality of life of small bowel recipients before and after transplantation and of home parenteral nutrition (HPN)-dependent patients before and after therapy. We examined quality of life across multiple areas of function including physical, social, and emotional indices. Methods The Quality of Life Instrument in the form of a self-administered questionnaire was completed voluntarily by the recipients of small intestinal transplants and by a cohort of HPN-dependent patients. Results Small intestinal transplant recipients reported significant improvement in the quality of their life and function. They also rated their quality of life and function during the pretransplant, TPN-dependent period to be worse than before the development of chronic intestinal failure. Similarly, HPN recipients reported significant worsening across most areas of quality of life when they compared their premorbid period to the HPN-dependent state. Conclusions TPN dependence causes significant impairment in the quality of life in most areas of functioning. In contrast, small intestinal transplantation restores the quality of life among recipients with functioning grafts.

Purpose: A clinical trial of intestinal transplantation (ltx) under tacrolimus and prednisone immunosuppression was initiated in June 1990 in children with irreversible intestinal failure and who were dependent on total parenteral nutrition (TPN). Methods: Fifty-five patients (28 girls, 27 boys) with a median age of 3.2 years (range, 0.5 to 18 years) received 58 intestinal transplants that included isolated small bowel (SB) (n = 17), liver SB (LSB) (n = 33), and multivisceral (MV) (n = 8) allografts. Nine patients also received bone marrow infusion, and there were 20 colonic allografts. Azathioprine, cyclophosphamide, or mycophenolate mofetil were used in different phases of the series. Indications for ltx included: gastroschisis (n = 14), volvulus (n = 13), necrotizing enterocolitis (n = 6), intestinal atresia (n = 8), chronic intestinal pseudoobstruction (n = 5), Hirschsprung's disease (n = 4), microvillus inclusion disease (n = 3), multiple polyposis (n = 1), and trauma (n = 1). Results: Currently, 30 patients are alive (patient survival, 55%; graft survival, 52%). Twenty-nine children with functioning grafts are living at home and off TPN, with a mean follow-up of 962 (range, 75 to 2,424) days. Immunologic complications have included liver allograft rejection (n = 18), intestinal allograft rejection (n = 52), posttransplant lymphoproliferative disease (n = 16), cytomegalovirus (n = 16) and graft-versus-host disease (n = 4). A combination of associated complications included intestinal perforation (n = 4), biliary leak (n = 3), bile duct stenosis (n = 1), intestinal leak (n = 6), dehiscence with evisceration (n = 4), hepatic artery thrombosis (n = 3), bleeding (n = 9), portal vein stenosis (n = 1), intraabdominal abscess (n = 11), and chylous ascites (n = 4). Graft loss occurred as a result of rejection (n = 8), infection (n = 12), technical complication (n = 8), and complications of TPN after graft removal (n = 3). There were four retransplants (SB, n=1; LSB n=3). Conclusions: Intestinal transplantation is a valid therapeutic option for patients with intestinal failure suffering complications of TPN. The complex clinical and immunologic course of these patients is reflected in a higher complication rate as well as patient and graft loss than seen after heart, liver, and kidney transplantation, although better than after lung transplantation.

We are gaining experience in small intestine transplantation, however, the procedure is still experimental. Morbidity and mortality can be significant with frequent rehospitalizations. The indications for small intestine transplantation are varied though most patients have developed short gut syndrome requiring total parenteral nutrition (TPN) for nutritional support. Patients may present with a chronic illness (such as Crohn's disease), chronic pain, and psychiatric comorbidity that may need to be addressed during the perioperative period. Faced with the complicated postoperative course, transplant recipients develop a range of endogenous and organic psychiatric disorders. Psychiatric treatment may be complicated by these factors in addition to the nutritional, biochemical, and metabolic abnormalities of a transplanted small intestine.

SummaryThere has been a great level of enthusiasm to downregulate overactive N-methyl-D-aspartate (NMDA) receptors to protect neurons from excitotoxicity. NMDA receptors play pivotal roles in basic brain development and functions as well as in neurological disorders and diseases. However, mechanistic understanding of antagonism in NMDA receptors is limited due to complete lack of antagonist-bound structures for the L-glutamate-binding GluN2 subunits. Here, we report the crystal structures of GluN1/GluN2A NMDA receptor ligand-binding domain (LBD) heterodimers in complex with GluN1- and GluN2-targeting antagonists. The crystal structures reveal that the antagonists, D-(−)-2-amino-5-phosphonopentanoic acid (D-AP5) and 1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA), have discrete binding modes and mechanisms for opening of the bilobed architecture of GluN2A LBD compared to the agonist-bound form. The current study shows distinct ways by which the conformations of NMDA receptor LBDs may be controlled and coupled to receptor inhibition and provides possible strategies to develop therapeutic compounds with higher subtype-specificity.Video Abstract