Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

Selective Cell-Surface Expression of Triheteromeric NMDA Receptors.

NMDA-type ionotropic glutamate receptors are critically involved in many brain functions and are implicated in a variety of brain disorders. Seven NMDA receptor subunits exist (GluN1, GluN2A-D, and GluN3A-B) that assemble into tetrameric receptor subtypes with distinct functional properties and physiological roles. The majority NMDA receptors are composed of two GluN1 and two GluN2 subunits, which can assemble into four diheteromeric receptors subtypes composed of GluN1 and one type of GluN2 subunit (e.g., GluN1/2A), and presumably also six triheteromeric receptor subtypes composed of GluN1 and two different GluN2 subunits (e.g., GluN1/2A/2B). Furthermore, the GluN1 subunit exists as eight splice variants (e.g., GluN1-1a and GluN1-1b isoforms), and two different GluN1 isoforms can co-assemble to also form triheteromeric NMDA receptors (e.g., GluN1-1a/1b/2A). Here, we describe a method to faithfully express triheteromeric NMDA receptors in heterologous expression systems by controlling the identity of two of the four subunits. This method overcomes the problem that co-expression of three different NMDA receptor subunits generates two distinct diheteromeric receptor subtypes as well as one triheteromeric receptor subtype, thereby confounding studies that require a homogenous population of triheteromeric NMDA receptors. The method has been applied to selectively express recombinant triheteromeric GluN1/2A/2B, GluN1/2A/2C, GluN1/2B/2D, GluN1-1a/GluN1-1b/2A, GluN1-1a/GluN1-1b/2B receptors with negligible co-expression of the respective diheteromeric receptor subtypes. This method therefore enables quantitative evaluation of functional and pharmacological properties of triheteromeric NMDA receptors, some of which are abundant NMDA receptor subtypes in the adult brain.

Molecular Dynamics Simulations of the Mutated Proton-Transferring a-Subunit of E. coli FoF1-ATP Synthase.

The membrane Fo factor of ATP synthase is highly sensitive to mutations in the proton half-channel leading to the functional blocking of the entire protein. To identify functionally important amino acids for the proton transport, we performed molecular dynamic simulations on the selected mutants of the membrane part of the bacterial FoF1-ATP synthase embedded in a native lipid bilayer: there were nine different mutations of a-subunit residues (aE219, aH245, aN214, aQ252) in the inlet half-channel. The structure proved to be stable to these mutations, although some of them (aH245Y and aQ252L) resulted in minor conformational changes. aH245 and aN214 were crucial for proton transport as they directly facilitated H+ transfer. The substitutions with nonpolar amino acids disrupted the transfer chain and water molecules or neighboring polar side chains could not replace them effectively. aE219 and aQ252 appeared not to be determinative for proton translocation, since an alternative pathway involving a chain of water molecules could compensate the ability of H+ transmembrane movement when they were substituted. Thus, mutations of conserved polar residues significantly affected hydration levels, leading to drastic changes in the occupancy and capacity of the structural water molecule clusters (W1-W3), up to their complete disappearance and consequently to the proton transfer chain disruption.

Rescuing tri-heteromeric NMDA receptor function: the potential of pregnenolone-sulfate in loss-of-function GRIN2B variants.

N-methyl-D-aspartate receptors (NMDARs emerging from GRIN genes) are tetrameric receptors that form diverse channel compositions in neurons, typically consisting of two GluN1 subunits combined with two GluN2(A-D) subunits. During prenatal stages, the predominant channels are di-heteromers with two GluN1 and two GluN2B subunits due to the high abundance of GluN2B subunits. Postnatally, the expression of GluN2A subunits increases, giving rise to additional subtypes, including GluN2A-containing di-heteromers and tri-heteromers with GluN1, GluN2A, and GluN2B subunits. The latter  emerge as the major receptor subtype at mature synapses in the hippocampus. Despite extensive research on purely di-heteromeric receptors containing two identical GRIN variants, the impact of a single variant on the function of other channel forms, notably tri-heteromers, is lagging. In this study, we systematically investigated the effects of two de novo GRIN2B variants (G689C and G689S) in pure, mixed di- and tri-heteromers. Our findings reveal that incorporating a single variant in mixed di-heteromers or tri-heteromers exerts a dominant negative effect on glutamate potency, although 'mixed' channels show improved potency compared to pure variant-containing di-heteromers. We show that a single variant within a receptor complex does not impair the response of all receptor subtypes to the positive allosteric modulator pregnenolone-sulfate (PS), whereas spermine completely fails to potentiate tri-heteromers containing GluN2A and -2B-subunits. We examined PS on primary cultured hippocampal neurons transfected with the variants, and observed a positive impact over current amplitudes and synaptic activity. Together, our study supports previous observations showing that mixed di-heteromers exhibit improved glutamate potency and extend these findings towards the exploration of the effect of Loss-of-Function variants over tri-heteromers. Notably, we provide an initial and crucial demonstration of the beneficial effects of GRIN2B-relevant potentiators on tri-heteromers. Our results underscore the significance of studying how different variants affect distinct receptor subtypes, as these effects cannot be inferred solely from observations made on pure di-heteromers. Overall, this study contributes to ongoing efforts to understand the pathophysiology of GRINopathies and provides insights into potential treatment strategies.

Molecular and physiological characterization of AIP1, encoding the acetolactate synthase regulatory subunit in rice.

Flooding deprives plants of oxygen and thereby causes severe stress by interfering with energy production, leading to growth retardation. Enzymes and metabolites may help protect plants from waterlogging and hypoxic environmental conditions. Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of branched-chain amino acids (BCAAs), providing the building blocks for proteins and various secondary metabolites. Additionally, under energy-poor conditions, free BCAAs can be used as an alternative energy source by mitochondria through a catabolic enzyme chain reaction. In this study, we characterized ALS-INTERACTING PROTEIN 1 (OsAIP1), which encodes the regulatory subunit of ALS in rice (Oryza sativa). This gene was expressed in all parts of the rice plant, and its expression level was significantly higher in submerged and low-oxygen environments. Rice transformants overexpressing OsAIP1 showed a higher survival rate under hypoxic stress than did non-transgenic control plants under the same conditions. The OsAIP1-overexpressing plants accumulated increased levels of BCAAs, demonstrating that OsAIP1 is an important factor in the hypoxia resistance mechanism. These results suggest that ALS proteins are part of a defense mechanism that improves the tolerance of plants to low-oxygen environments.

Alternative splicing for tuneable expression of protein subunits at desired ratios.

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

Coregulation of NDC80 Complex Subunits Determines the Fidelity of the Spindle-Assembly Checkpoint and Mitosis.

NDC80 complex (NDC80C) is composed of four subunits (SPC24, SPC25, NDC80, and NUF2) and is vital for kinetochore-microtubule (KT-MT) attachment during mitosis. Paradoxically, NDC80C also functions in the activation of the spindle-assembly checkpoint (SAC). This raises an interesting question regarding how mitosis is regulated when NDC80C levels are compromised. Using a degron-mediated depletion system, we found that acute silencing of SPC24 triggered a transient mitotic arrest followed by mitotic slippage. SPC24-deficient cells were unable to sustain SAC activation despite the loss of KT-MT interaction. Intriguingly, our results revealed that other subunits of the NDC80C were co-downregulated with SPC24 at a posttranslational level. Silencing any individual subunit of NDC80C likewise reduced the expression of the entire complex. We found that the SPC24-SPC25 and NDC80-NUF2 subcomplexes could be individually stabilized using ectopically expressed subunits. The synergism of SPC24 downregulation with drugs that promote either mitotic arrest or mitotic slippage further underscored the dual roles of NDC80C in KT-MT interaction and SAC maintenance. The tight coordinated regulation of NDC80C subunits suggests that targeting individual subunits could disrupt mitotic progression and provide new avenues for therapeutic intervention. IMPLICATIONS: These results highlight the tight coordinated regulation of NDC80C subunits and their potential as targets for antimitotic therapies.

Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function.

The sixth family phosphodiesterases (PDE6) are principal effector enzymes of the phototransduction cascade in rods and cones. Maturation of nascent PDE6 protein into a functional enzyme relies on a coordinated action of ubiquitous chaperone HSP90, its specialized cochaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and the regulatory Pγ-subunit of PDE6. Deficits in PDE6 maturation and function underlie severe visual disorders and blindness. Here, to elucidate the roles of HSP90, AIPL1, and Pγ in the maturation process, we developed the heterologous expression system of human cone PDE6C in insect cells allowing characterization of the purified enzyme. We demonstrate that in the absence of Pγ, HSP90, and AIPL1 convert the inactive and aggregating PDE6C species into dimeric PDE6C that is predominantly misassembled. Nonetheless, a small fraction of PDE6C is properly assembled and fully functional. From the analysis of mutant mice that lack both rod Pγ and PDE6C, we conclude that, in contrast to the cone enzyme, no maturation of rod PDE6AB occurs in the absence of Pγ. Co-expression of PDE6C with AIPL1 and Pγ in insect cells leads to a fully mature enzyme that is equivalent to retinal PDE6. Lastly, using immature PDE6C and purified chaperone components, we reconstituted the process of the client maturation in vitro. Based on this analysis we propose a scheme for the PDE6 maturation process.

G Protein Subunit Beta 3 (GNB3) Variant Is Associated with Biochemical Changes in Brazilian Patients with Hypertension. / Variante da Subunidade Beta 3 da Proteína G ( GNB3 ) Está Associada a Alterações Bioquímicas em Pacientes Brasileiros com Hipertensão.

BACKGROUND: Central Illustration : G Protein Subunit Beta 3 (GNB3) Variant Is Associated with Biochemical Changes in Brazilian Patients with Hypertension. BACKGROUND: Genes and their variants associated with environmental factors contribute to the development of the hypertensive phenotype. The G protein beta 3 subunit gene (GNB3) is involved in the intracellular signaling process, and its variants have been related to susceptibility to arterial hypertension. OBJECTIVE: To determine the association of the GNB3 variant (rs5443:C>T) with arterial hypertension, biochemical parameters, age, and obesity in hypertensive and normotensive individuals from Ouro Preto, Minas Gerais, Brazil. METHOD: The identification of variants was performed by real-time PCR, using the TaqMan® system, in 310 samples (155 hypertensive and 155 normotensive). Biochemical analyses (renal function, lipid profile and glycemia) were performed from the serum using UV/Vis spectrophotometry and ion-selective electrode. A multiple logistic regression model was used to identify factors associated with arterial hypertension. The analysis of continuous variables with normal distribution was performed using the unpaired Student's t test; non-normal data were analyzed using Mann-Whitney. P < 0.05 was considered significant. RESULTS: The rs5443:C>T variant was not associated with arterial hypertension in the evaluated population (p = 0.88). Regarding biochemical measures, the T allele was associated with high levels of triglycerides, glucose and uric acid in hypertensive individuals (p < 0.05). CONCLUSION: These results show the importance of genetic diagnosis to prevent the causes and consequences of diseases and imply that the GNB3 rs5443:C>T variant may be associated with changes in the biochemical profile in hypertensive individuals.

Single-Nucleotide Polymorphism in Genes Encoding G Protein Subunits GNB3 and GNAQ Increase the Risk of Cardiovascular Morbidity among Patients Undergoing Renal Replacement Therapy.

Single-nucleotide polymorphisms in G protein subunits are linked to an increased risk of cardiovascular events among the general population. We assessed the effects of GNB3 c.825C > T, GNAQ -695/-694GC > TT, and GNAS c.393C > T polymorphisms on the risk of cardiovascular events among 454 patients undergoing renal replacement therapy. The patients were followed up for a median of 4.5 years after the initiation of dialysis. Carriers of the TT/TT genotype of GNAQ required stenting because of coronary artery stenosis (p = 0.0009) and developed cardiovascular events involving more than one organ system (p = 0.03) significantly earlier and more frequently than did the GC/TT or GC/GC genotypes. Multivariate analysis found that the TT/TT genotype of GNAQ was an independent risk factor for coronary artery stenosis requiring stent (hazard ratio, 4.5; p = 0.001), cardiovascular events (hazard ratio, 1.93; p = 0.04) and cardiovascular events affecting multiple organs (hazard ratio, 4.9; p = 0.03). In the subgroup of male patients left ventricular dilatation with abnormally increased LVEDD values occurred significantly more frequently in TT genotypes of GNB3 than in CT/CC genotypes (p = 0.007). Our findings suggest that male dialysis patients carrying the TT genotype of GNB3 are at higher risk of left ventricular dilatation and that dialysis patients carrying the TT/TT genotype of GNAQ are prone to coronary artery stenosis and severe cardiovascular events.

Differential regulation of tetramerization of the AMPA receptor glutamate-gated ion channel by auxiliary subunits.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) auxiliary subunits are specialized, nontransient binding partners of AMPARs that modulate AMPAR channel gating properties and pharmacology, as well as their biogenesis and trafficking. The most well-characterized families of auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs), cornichon homologs (CNIHs), and the more recently discovered GSG1-L. These auxiliary subunits can promote or reduce surface expression of AMPARs (composed of GluA1-4 subunits) in neurons, thereby impacting their functional role in membrane signaling. Here, we show that CNIH-2 enhances the tetramerization of WT and mutant AMPARs, presumably by increasing the overall stability of the tetrameric complex, an effect that is mainly mediated by interactions with the transmembrane domain of the receptor. We also find CNIH-2 and CNIH-3 show receptor subunit-specific actions in this regard with CNIH-2 enhancing both GluA1 and GluA2 tetramerization, whereas CNIH-3 only weakly enhances GluA1 tetramerization. These results are consistent with the proposed role of CNIHs as endoplasmic reticulum cargo transporters for AMPARs. In contrast, TARP γ-2, TARP γ-8, and GSG1-L have no or negligible effect on AMPAR tetramerization. On the other hand, TARP γ-2 can enhance receptor tetramerization but only when directly fused with the receptor at a maximal stoichiometry. Notably, surface expression of functional AMPARs was enhanced by CNIH-2 to a greater extent than TARP γ-2, suggesting that this distinction aids in maturation and membrane expression. These experiments define a functional distinction between CNIHs and other auxiliary subunits in the regulation of AMPAR biogenesis.

Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal subunit regulation of the cardiac voltage-gated sodium channel.

Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory ß-subunits. The ß-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous ß-subunits. To better reveal ß-subunit functions, we engineered a human cell line devoid of endogenous NaV ß-subunits and their immediate phylogenetic relatives. This new cell line, ß-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the ß-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac NaV α-subunit, NaV1.5, was highly sensitive to ß-subunit modulation and revealed that each ß-subunit and even MPZ imparted unique gating properties. Furthermore, combining ß1 and ß2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of ß-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other α/ß NaV complexes.

RNA Pol II preferentially regulates ribosomal protein expression by trapping disassociated subunits.

RNA polymerase II (RNA Pol II) has been recognized as a passively regulated multi-subunit holoenzyme. However, the extent to which RNA Pol II subunits might be important beyond the RNA Pol II complex remains unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, i.e., "specific degradation of disassociated subunits (SDDS)," we demonstrated that dRPB3 functions as a regulatory component of RNA Pol II to enable the preferential control of 3' end processing of ribosomal protein genes directly through its N-terminal domain. Machine learning analysis of large-scale genomic features revealed that the little elongation complex (LEC) helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3' end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that RNA Pol II gains specific regulatory functions by trapping disassociated subunits in mammalian cells.

Quantitative analysis of NMDA receptor subunits proteins in mouse brain.

NMDA-type glutamate receptors (NMDARs) are tetrameric channel complex composed of two subunits of GluN1, which is encoded by a single gene and diversified by alternative splicing, and two subunits from four subtypes of GluN2, leading to various combinations of subunits and channel specificities. However, there is no comprehensive quantitative analysis of GluN subunit proteins for relative comparison, and their compositional ratios at various regions and developmental stages have not been clarified. Here we prepared six chimeric subunits, by fusing an N-terminal side of the GluA1 subunit with a C-terminal side of each of two splicing isoforms of GluN1 subunit and four GluN2 subunits, with which titers of respective NMDAR subunit antibodies could be standardized using common GluA1 antibody, thus enabling quantification of relative protein levels of each NMDAR subunit by western blotting. We determined relative protein amounts of NMDAR subunits in crude, membrane (P2) and microsomal fractions prepared from the cerebral cortex, hippocampus and cerebellum in adult mice. We also examined amount changes in the three brain regions during developmental stages. Their relative amounts in the cortical crude fraction were almost parallel to those of mRNA expression, except for some subunits. Interestingly, a considerable amount of GluN2D protein existed in adult brains, although its transcription level declines after early postnatal stages. GluN1 was larger in quantity than GluN2 in the crude fraction, whereas GluN2 increased in the membrane component-enriched P2 fraction, except in the cerebellum. These data will provide the basic spatio-temporal information on the amount and composition of NMDARs.

Pathophysiological Roles of Auxiliary Calcium Channel α2δ Subunits.

α2δ proteins serve as auxiliary subunits of voltage-gated calcium channels, which are essential components of excitable cells such as skeletal and heart muscles, nerve cells of the brain and the peripheral nervous system, as well as endocrine cells. Over the recent years, α2δ proteins have been identified as critical regulators of synaptic functions, including the formation and differentiation of synapses. These functions require signalling mechanisms which are partly independent of calcium channels. Hence, in light of these features it is not surprising that the genes encoding for the four α2δ isoforms have recently been linked to neurological and neurodevelopmental disorders including epilepsy, autism spectrum disorders, schizophrenia, and depressive and bipolar disorders. Despite the increasing number of identified disease-associated mutations, the underlying pathophysiological mechanisms are only beginning to emerge. However, a thorough understanding of the pathophysiological role of α2δ proteins ideally serves two purposes: first, it will contribute to our understanding of general pathological mechanisms in synaptic disorders. Second, it may support the future development of novel and specific treatments for brain disorders. In this context, it is noteworthy that the antiepileptic and anti-allodynic drugs gabapentin and pregabalin both act via binding to α2δ proteins and are among the top sold drugs for treating neuropathic pain. In this book chapter, we will discuss recent developments in our understanding of the functions of α2δ proteins, both as calcium channel subunits and as independent regulatory entities. Furthermore, we present and summarize recently identified and likely pathogenic mutations in the genes encoding α2δ proteins and discuss potential underlying pathophysiological consequences at the molecular and structural level.

Elongator and the role of its subcomplexes in human diseases.

The Elongator complex was initially identified in yeast, and a variety of distinct cellular functions have been assigned to the complex. In the last decade, several research groups focussed on dissecting its structure, tRNA modification activity and role in translation regulation. Recently, Elongator emerged as a crucial factor for various human diseases, and its involvement has triggered a strong interest in the complex from numerous clinical groups. The Elongator complex is highly conserved among eukaryotes, with all six subunits (Elp1-6) contributing to its stability and function. Yet, recent studies have shown that the two subcomplexes, namely the catalytic Elp123 and accessory Elp456, may have distinct roles in the development of different neuronal subtypes. This Commentary aims to provide a brief overview and new perspectives for more systematic efforts to explore the functions of the Elongator in health and disease.

hERG1 channel subunit composition mediates proton inhibition of rapid delayed rectifier potassium current (IKr) in cardiomyocytes derived from hiPSCs.

The voltage-gated channel, hERG1, conducts the rapid delayed rectifier potassium current (IKr) and is critical for human cardiac repolarization. Reduced IKr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden death. At least two subunits form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG1a/1b abundance modulate IKr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue suggest that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on IKr and its response to external stressors is not well understood. Here, we used a substrate-driven human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturation model to investigate how changes in relative hERG1a/1b subunit abundance impact the response of native IKr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. IKr recorded from immatured hiPSC-CMs displays a 2-fold greater inhibition by extracellular acidosis (pH 6.3) compared with matured hiPSC-CMs. Quantitative RT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared with immatured hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by increased IKr. Silencing hERG1b's impact on native IKr kinetics by overexpressing a polypeptide identical to the hERG1a N-terminal Per-Arnt-Sim domain reduced the magnitude of IKr proton inhibition in immatured hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and determines IKr proton sensitivity in hiPSC-CMs.

Addition of Psathyrostachys huashanica HMW glutenin subunit expresses positive contribution to protein polymerization and gluten microstructure of receptor wheat.

Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) is considered as a valuable wild germplasm for wheat improvement on account of its numerous outstanding traits. During this study, 7182-1Ns with higher quality was screened out, a series of experiments were conducted to clarify the reasons of quality improvement. The results indicated 7182-1Ns was carried a novel high-molecular-weight glutenin subunit (HMW-GS) from P. huashanica, designated as P. huashanica' subunit in wheat (HS), which changed the HMW-GS compositions, increased the proportion of glutenins in wheat gluten protein, accelerated the accumulation speed of unextractable polymeric protein (UPP) during grain development stage accelerated, and a denser microstructure of the gluten network was formed in the dough. Therefore, the current research provides important reference on effectively utilize 7182-1Ns as an intermediate germplasm for quality breeding improvement.

NMDA Receptor C-Terminal Domain Signalling in Development, Maturity, and Disease.

The NMDA receptor is a Ca2+-permeant glutamate receptor which plays key roles in health and disease. Canonical NMDARs contain two GluN2 subunits, of which 2A and 2B are predominant in the forebrain. Moreover, the relative contribution of 2A vs. 2B is controlled both developmentally and in an activity-dependent manner. The GluN2 subtype influences the biophysical properties of the receptor through difference in their N-terminal extracellular domain and transmembrane regions, but they also have large cytoplasmic Carboxyl (C)-terminal domains (CTDs) which have diverged substantially during evolution. While the CTD identity does not influence NMDAR subunit specific channel properties, it determines the nature of CTD-associated signalling molecules and has been implicated in mediating the control of subunit composition (2A vs. 2B) at the synapse. Historically, much of the research into the differential function of GluN2 CTDs has been conducted in vitro by over-expressing mutant subunits, but more recently, the generation of knock-in (KI) mouse models have allowed CTD function to be probed in vivo and in ex vivo systems without heterologous expression of GluN2 mutants. In some instances, findings involving KI mice have been in disagreement with models that were proposed based on earlier approaches. This review will examine the current research with the aim of addressing these controversies and how methodology may contribute to differences between studies. We will also discuss the outstanding questions regarding the role of GluN2 CTD sequences in regulating NMDAR subunit composition, as well as their relevance to neurodegenerative disease and neurodevelopmental disorders.