Determinants, maintenance, and function of organellar pH.

The protonation state of soluble and membrane-associated macromolecules dictates their charge, conformation, and functional activity. In addition, protons (H+ or their equivalents) partake in numerous metabolic reactions and serve as a source of electrochemical energy to drive the transmembrane transport of both organic and inorganic substrates. Stringent regulation of the intracellular pH is therefore paramount to homeostasis. Although the regulation of the cytosolic pH has been studied extensively, our understanding of the determinants of the H+ concentration ([H+]) of intracellular organelles has developed more slowly, limited by their small size and inaccessibility. Recently, however, targeting of molecular probes to the organellar lumen together with advances in genomic, proteomic, and electrophysiological techniques have led to the identification and characterization of unique pumps, channels, and transporters responsible for the establishment and maintenance of intraorganellar pH. These developments and their implications for cellular function in health and disease are the subject of this review.

Roles of Endomembrane Alkali Cation/Proton Exchangers in Synaptic Function and Neurodevelopmental Disorders.

Endomembrane alkali cation (Na+, K+)/proton (H+) exchangers (eNHEs) are increasingly associated with neurological disorders. These eNHEs play integral roles in regulating the luminal pH, processing, and trafficking of cargo along the secretory (Golgi and post-Golgi vesicles) and endocytic (early, recycling, and late endosomes) pathways, essential regulatory processes vital for neuronal development and plasticity. Given the complex morphology and compartmentalization of multipolar neurons, the contribution of eNHEs in maintaining optimal pH homeostasis and cargo trafficking is especially significant during periods of structural and functional development and remodeling. While the importance of eNHEs has been demonstrated in a variety of non-neuronal cell types, their involvement in neuronal function is less well understood. In this review, we will discuss their emerging roles in excitatory synaptic function, particularly as it pertains to cellular learning and remodeling. We will also explore their connections to neurodevelopmental conditions, including intellectual disability, autism, and attention deficit hyperactivity disorders.

Structural basis of autoinhibition of the human NHE3-CHP1 complex.

Sodium-proton exchanger 3 (NHE3/SLC9A3) located in the apical membrane of renal and gastrointestinal epithelia mediates salt and fluid absorption and regulates pH homeostasis. As an auxiliary regulatory factor of NHE proteins, calcineurin B homologous protein 1 (CHP1) facilitates NHE3 maturation, plasmalemmal expression, and pH sensitivity. Dysfunctions of NHE3 are associated with renal and digestive system disorders. Here, we report the cryo-electron microscopy structure of the human NHE3-CHP1 complex in its inward-facing conformation. We found that a cytosolic helix-loop-helix motif in NHE3 blocks the intracellular cavity formed between the core and dimerization domains, functioning as an autoinhibitory element and hindering substrate transport. Furthermore, two phosphatidylinositol molecules are found to bind to the peripheric juxtamembrane sides of the complex, function as anchors to stabilize the complex, and may thus enhance its transport activity.

Sleep and daytime behavior in individuals with Christianson Syndrome.

BACKGROUND: The objectives of this study were to: 1) characterize the sleep behaviors and symptoms of individuals with Christianson Syndrome (CS) by means of validated questionnaires; and 2) determine their associations with daytime emotional and behavioral symptoms in this population. METHODS: Participants included 16 boys genetically diagnosed with CS, between 2.5 and 40 years of age (M = 14.5 ± 8.08). Parents completed questionnaires regarding the sleep, daytime behavior, and health of their child. RESULTS: Of the participants, 31% did not obtain the recommended amount of sleep for their age, 43% experienced a prolonged sleep latency, and 88% had a clinical or sub-clinical score for at least one subscale of the Sleep Disturbance Scale for Children (SDSC). Specific problems detected included insomnia, sleep-wake transition disorders, periodic limb movements in sleep, and sleep related breathing disorders. About half of the participants manifested emotional and behavioral problems at clinical levels. Higher levels of sleep disturbances were associated with higher levels of behavioral and emotional daytime symptoms. CONCLUSIONS: Sleep problems are common in individuals with CS and are associated with daytime behavioral and emotional symptoms.

Structure and mechanism of the human NHE1-CHP1 complex.

Sodium/proton exchanger 1 (NHE1) is an electroneutral secondary active transporter present on the plasma membrane of most mammalian cells and plays critical roles in regulating intracellular pH and volume homeostasis. Calcineurin B-homologous protein 1 (CHP1) is an obligate binding partner that promotes NHE1 biosynthetic maturation, cell surface expression and pH-sensitivity. Dysfunctions of either protein are associated with neurological disorders. Here, we elucidate structures of the human NHE1-CHP1 complex in both inward- and inhibitor (cariporide)-bound outward-facing conformations. We find that NHE1 assembles as a symmetrical homodimer, with each subunit undergoing an elevator-like conformational change during cation exchange. The cryo-EM map reveals the binding site for the NHE1 inhibitor cariporide, illustrating how inhibitors block transport activity. The CHP1 molecule differentially associates with these two conformational states of each NHE1 monomer, and this association difference probably underlies the regulation of NHE1 pH-sensitivity by CHP1.

Loss of SLC9A6/NHE6 impairs nociception in a mouse model of Christianson syndrome.

Children diagnosed with Christianson syndrome (CS), a rare X-linked neurodevelopmental disorder characterized by intellectual disability, epilepsy, ataxia, and mutism, also suffer from hyposensitivity to pain. This places them at risk of sustaining serious injuries that often go unattended. Christianson syndrome is caused by mutations in the alkali cation/proton exchanger SLC9A6/NHE6 that regulates recycling endosomal pH homeostasis and trafficking. Yet, it remains unclear how defects in this transporter lead to altered somatosensory functions. In this study, we validated a Nhe6 knockout (KO) mouse as a model of CS and used it to identify the cellular mechanisms underlying the elevated pain tolerance observed in CS patients. Within the central nervous system, NHE6 immunolabelling is detected in a small percentage of cortical neurons involved in pain processing, including those within the primary somatosensory and the anterior cingulate cortices as well as the periaqueductal gray. Interestingly, it is expressed in a larger percentage of nociceptors. Behaviourally, Nhe6 KO mice have decreased nocifensive responses to acute noxious thermal, mechanical, and chemical (ie, capsaicin) stimuli. The reduced capsaicin sensitivity in the KO mice correlates with a decreased expression of the transient receptor potential channel TRPV1 at the plasma membrane and capsaicin-induced Ca influx in primary cultures of nociceptors. These data indicate that NHE6 is a significant determinant of nociceptor function and pain behaviours, vital sensory processes that are impaired in CS.

Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome.

Genetic screening has identified numerous variants of the endosomal solute carrier family 9 member A6 (SLC9A6)/(Na+,K+)/H+ exchanger 6 (NHE6) gene that cause Christianson syndrome, a debilitating X-linked developmental disorder associated with a range of neurological, somatic, and behavioral symptoms. Many of these variants cause complete loss of NHE6 expression, but how subtler missense substitutions or nonsense mutations that partially truncate its C-terminal cytoplasmic regulatory domain impair NHE6 activity and endosomal function are poorly understood. Here, we describe the molecular and cellular consequences of six unique mutations located in the N-terminal cytoplasmic segment (A9S), the membrane ion translocation domain (L188P and G383D), and the C-terminal regulatory domain (E547*, R568Q, and W570*) of human NHE6 that purportedly cause disease. Using a heterologous NHE6-deficient cell expression system, we show that the biochemical, catalytic, and cellular properties of the A9S and R568Q variants were largely indistinguishable from those of the WT transporter, which obscured their disease significance. By contrast, the L188P, G383D, E547*, and W570* mutants exhibited variable deficiencies in biosynthetic post-translational maturation, membrane sorting, pH homeostasis in recycling endosomes, and cargo trafficking, and they also triggered apoptosis. These findings broaden our understanding of the molecular dysfunctions of distinct NHE6 variants associated with Christianson syndrome.

Paxillin S273 Phosphorylation Regulates Adhesion Dynamics and Cell Migration through a Common Protein Complex with PAK1 and ßPIX.

Cell migration is an important biological phenomenon involved in many homeostatic and aberrant physiological processes. Phosphorylation of the focal adhesion adaptor protein, paxillin, on serine 273 (S273) has been implicated as a key regulator of cell migration. Here, it is shown that phosphorylation on paxillin S273 leads to highly migratory cells with small dynamic adhesions. Adhesions at protrusive edges of the cell were more dynamic than adhesions at retracting edges. Temporal image correlation microscopy revealed that these dynamic adhesions undergo rapid binding of paxillin, PAK1 and ßPIX. We identified membrane proximal adhesion subdomains in protrusive regions of the cell that show rapid protein binding that is dependent on paxillin S273 phosphorylation, PAK1 kinase activity and phosphatases. These dynamic adhesion subdomains corresponded to regions of the adhesion that also show co-binding of paxillin/PAK1 and paxillin/ßPIX complexes. It is likely that parts of individual adhesions are more dynamic while others are less dynamic due to their association with the actin cytoskeleton. Variable adhesion and binding dynamics are regulated via differential paxillin S273 phosphorylation across the cell and within adhesions and are required for regulated cell migration. Dysregulation through phosphomutants, PAK1-KD or ßPIX mutants resulted in large stable adhesions, long protein binding times and slow cell migration. Dysregulation through phosphomimics or PAK1-CA led to small dynamic adhesions and rapid cell migration reminiscent of highly migratory cancer cells. Thus, phosphorylation of paxillin S273 is a key regulator of cell migration through recruitment of ßPIX and PAK1 to sites of adhesion.

A Christianson syndrome-linked deletion mutation (Δ287ES288) in SLC9A6 impairs hippocampal neuronal plasticity.

Christianson Syndrome is a rare but increasingly diagnosed X-linked intellectual disability disorder that arises from mutations in SLC9A6/NHE6, a pH-regulating transporter that localizes to early and recycling endosomes. We have recently reported that one of the originally identified disease-causing mutations in NHE6 (p.E287-S288del, or ΔES) resulted in a loss of its pH regulatory function. However, the impact of this mutation upon neuronal synapse formation and plasticity is unknown. Here, we investigate the consequences of the ΔES mutant upon mouse hippocampal pyramidal neurons by expressing a fluorescently-labeled ΔES NHE6 construct into primary hippocampal neurons. Neurons expressing the ΔES mutant showed significant reductions in mature dendritic spine density with a concurrent increase in immature filopodia. Furthermore, compared to wild-type (WT), ΔES-containing endosomes are redirected away from early and recycling endosomes toward lysosomes. In parallel, the ΔES mutant reduced the trafficking of glutamatergic AMPA receptors to excitatory synapses and increased their accumulation within lysosomes for potential degradation. Upon long-term potentiation (LTP), neurons expressing ΔES failed to undergo significant structural and functional changes as observed in controls and WT transfectants. Interestingly, synapse density and LTP-induced synaptic remodeling in ΔES-expressing neurons were partially restored by bafilomycin, a vesicular alkalinisation agent, or by leupeptin, an inhibitor of lysosomal proteolytic degradation. Overall, our results demonstrate that the ∆ES mutation attenuates synapse density and structural and functional plasticity in hippocampal neurons. These deficits may be partially due to the mistargeting of AMPA receptors and other cargos to lysosomes, thereby preventing their trafficking during synaptic remodeling. This mechanism may contribute to the cognitive learning deficits observed in patients with Christianson Syndrome and suggests a potential therapeutic strategy for treatment.

Assessing Variation in US Soybean Seed Composition (Protein and Oil).

Soybean [Glycine max (L.) Merr.] seed composition and yield are a function of genetics (G), environment (E), and management (M) practices, but contribution of each factor to seed composition and yield are not well understood. The goal of this synthesis-analysis was to identify the main effects of G, E, and M factors on seed composition (protein and oil concentration) and yield. The entire dataset (13,574 data points) consisted of 21 studies conducted across the United States (US) between 2002 and 2017 with varying treatments and all reporting seed yield and composition. Environment (E), defined as site-year, was the dominant factor accounting for more than 70% of the variation for both seed composition and yield. Of the crop management factors: (i) delayed planting date decreased oil concentration by 0.007 to 0.06% per delayed week (R 2∼0.70) and a 0.01 to 0.04 Mg ha-1 decline in seed yield per week, mainly in northern latitudes (40-45 N); (ii) crop rotation (corn-soybean) resulted in an overall positive impact for both seed composition and yield (1.60 Mg ha-1 positive yield difference relative to continuous soybean); and (iii) other management practices such as no-till, seed treatment, foliar nutrient application, and fungicide showed mixed results. Fertilizer N application in lower quantities (10-50 kg N ha-1) increased both oil and protein concentration, but seed yield was improved with rates above 100 kg N ha-1. At southern latitudes (30-35 N), trends of reduction in oil and increases in protein concentrations with later maturity groups (MG, from 3 to 7) was found. Continuing coordinated research is critical to advance our understanding of G × E × M interactions.

A potential gain-of-function variant of SLC9A6 leads to endosomal alkalinization and neuronal atrophy associated with Christianson Syndrome.

Loss-of-function mutations in the recycling endosomal (Na+,K+)/H+ exchanger gene SLC9A6/NHE6 result in overacidification and dysfunction of endosomal-lysosomal compartments, and cause a neurodevelopmental and degenerative form of X-linked intellectual disability called Christianson Syndrome (CS). However, knowledge of the disease heterogeneity of CS is limited. Here, we describe the clinical features and underlying molecular and cellular mechanisms associated with a CS patient carrying a de novo missense variant (p.Gly218Arg; G218R) of a conserved residue in its ion translocation domain that results in a potential gain-of-function. The patient manifested several core symptoms typical of CS, including pronounced cognitive impairment, mutism, epilepsy, ataxia and microcephaly; however, deterioration of motor function often observed after the first decade of life in CS children with total loss of SLC9A6/NHE6 function was not evident. In transfected non-neuronal cells, complex glycosylation and half-life of the G218R were significantly decreased compared to the wild-type transporter. This correlated with elevated ubiquitination and partial proteasomal-mediated proteolysis of G218R. However, a major fraction was delivered to the plasma membrane and endocytic pathways. Compared to wild-type, G218R-containing endosomes were atypically alkaline and showed impaired uptake of recycling endosomal cargo. Moreover, instead of accumulating in recycling endosomes, G218R was redirected to multivesicular bodies/late endosomes and ejected extracellularly in exosomes rather than progressing to lysosomes for degradation. Attenuated acidification and trafficking of G218R-containing endosomes were also observed in transfected hippocampal neurons, and correlated with diminished dendritic branching and density of mature mushroom-shaped spines and increased appearance of filopodia-like protrusions. Collectively, these findings expand our understanding of the genetic diversity of CS and further elucidate a critical role for SLC9A6/NHE6 in fine-tuning recycling endosomal pH and cargo trafficking, processes crucial for the maintenance of neuronal polarity and mature synaptic structures.

A recurrent missense variant in SLC9A7 causes nonsyndromic X-linked intellectual disability with alteration of Golgi acidification and aberrant glycosylation.

We report two unrelated families with multigenerational nonsyndromic intellectual disability (ID) segregating with a recurrent de novo missense variant (c.1543C>T:p.Leu515Phe) in the alkali cation/proton exchanger gene SLC9A7 (also commonly referred to as NHE7). SLC9A7 is located on human X chromosome at Xp11.3 and has not yet been associated with a human phenotype. The gene is widely transcribed, but especially abundant in brain, skeletal muscle and various secretory tissues. Within cells, SLC9A7 resides in the Golgi apparatus, with prominent enrichment in the trans-Golgi network (TGN) and post-Golgi vesicles. In transfected Chinese hamster ovary AP-1 cells, the Leu515Phe mutant protein was correctly targeted to the TGN/post-Golgi vesicles, but its N-linked oligosaccharide maturation as well as that of a co-transfected secretory membrane glycoprotein, vesicular stomatitis virus G (VSVG) glycoprotein, was reduced compared to cells co-expressing SLC9A7 wild-type and VSVG. This correlated with alkalinization of the TGN/post-Golgi compartments, suggestive of a gain-of-function. Membrane trafficking of glycosylation-deficient Leu515Phe and co-transfected VSVG to the cell surface, however, was relatively unaffected. Mass spectrometry analysis of patient sera also revealed an abnormal N-glycosylation profile for transferrin, a clinical diagnostic marker for congenital disorders of glycosylation. These data implicate a crucial role for SLC9A7 in the regulation of TGN/post-Golgi pH homeostasis and glycosylation of exported cargo, which may underlie the cellular pathophysiology and neurodevelopmental deficits associated with this particular nonsyndromic form of X-linked ID.

A major natural genetic variation associated with root system architecture and plasticity improves waterlogging tolerance and yield in soybean.

Natural genetic variations in waterlogging tolerance are controlled by multiple genes mapped as quantitative trait loci (QTLs) in major crops, including soybean (Glycine max L.). In this research, 2 novel QTLs associated with waterlogging tolerance were mapped from an elite/exotic soybean cross. The subsequent research was focused on a major QTL (qWT_Gm03) with the tolerant allele from the exotic parent. This QTL was isolated into near-isogenic backgrounds, and its effects on waterlogging tolerance were validated in multiple environments. Fine mapping narrowed qWT_Gm03 into a genomic region of <380 Kbp excluding Rps1 gene for Phytophthora sojae resistance. The tolerant allele of qWT_Gm03 promotes root growth under nonstress conditions and favourable root plasticity under waterlogging, resulting in improved waterlogging tolerance, yield, and drought tolerance-related traits, possibly through more efficient water/nutrient uptakes. Meanwhile, involvement of auxin pathways was also identified in the regulation of waterlogging tolerance, as the genotypic differences of qWT_Gm03 in waterlogging tolerance and formation of adventitious/aerial roots can be complemented by an exogenous auxin-biosynthesis inhibitor. These findings provided genetic resources to address the urgent demand of improving waterlogging tolerance in soybean and revealed the determinant roles of root architecture and plasticity in the plant adaptation to waterlogging.

SMAD5 signaling: more than meets the nuclei.

SMADs are essential transcriptional effectors of transforming growth factor-ß (TGFß)/TGFß-related signaling that underlies embryonic development and adult homeostasis. A recent study by Fang et al. in Cell Research adds to this biological complexity by demonstrating an atypical cytoplasmic role for SMAD5 in modulating the bioenergetic homeostasis (i.e., glycolysis and mitochondrial respiration) of cells in response to fluctuations in intracellular pH that is independent of receptor signaling.

A Christianson syndrome-linked deletion mutation (∆(287)ES(288)) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death.

BACKGROUND: Christianson Syndrome, a recently identified X-linked neurodevelopmental disorder, is caused by mutations in the human gene SLC9A6 encoding the recycling endosomal alkali cation/proton exchanger NHE6. The patients have pronounced limitations in cognitive ability, motor skills and adaptive behaviour. However, the mechanistic basis for this disorder is poorly understood as few of the more than 20 mutations identified thus far have been studied in detail. METHODS: Here, we examined the molecular and cellular consequences of a 6 base-pair deletion of amino acids Glu(287) and Ser(288) (∆ES) in the predicted seventh transmembrane helix of human NHE6 expressed in established cell lines (CHO/AP-1, HeLa and neuroblastoma SH-SY5Y) and primary cultures of mouse hippocampal neurons by measuring levels of protein expression, stability, membrane trafficking, endosomal function and cell viability. RESULTS: In the cell lines, immunoblot analyses showed that the nascent mutant protein was properly synthesized and assembled as a homodimer, but its oligosaccharide maturation and half-life were markedly reduced compared to wild-type (WT) and correlated with enhanced ubiquitination leading to both proteasomal and lysosomal degradation. Despite this instability, a measurable fraction of the transporter was correctly sorted to the plasma membrane. However, the rates of clathrin-mediated endocytosis of the ∆ES mutant as well as uptake of companion vesicular cargo, such as the ligand-bound transferrin receptor, were significantly reduced and correlated with excessive endosomal acidification. Notably, ectopic expression of ∆ES but not WT induced apoptosis when examined in AP-1 cells. Similarly, in transfected primary cultures of mouse hippocampal neurons, membrane trafficking of the ∆ES mutant was impaired and elicited marked reductions in total dendritic length, area and arborization, and triggered apoptotic cell death. CONCLUSIONS: These results suggest that loss-of-function mutations in NHE6 disrupt recycling endosomal function and trafficking of cargo which ultimately leads to neuronal degeneration and cell death in Christianson Syndrome.

Determinants of Cation Permeation and Drug Sensitivity in Predicted Transmembrane Helix 9 and Adjoining Exofacial Re-entrant Loop 5 of Na+/H+ Exchanger NHE1.

Mammalian Na(+)/H(+) exchangers (NHEs) regulate numerous physiological processes and are involved in the pathogenesis of several diseases, including tissue ischemia and reperfusion injuries, cardiac hypertrophy and failure, and cancer progression. Hence, NHEs are being targeted for pharmaceutical-based clinical therapies, but pertinent information regarding the structural elements involved in cation translocation and drug binding remains incomplete. Molecular manipulations of the prototypical NHE1 isoform have implicated several predicted membrane-spanning (M) helices, most notably M4, M9, and M11, as important determinants of cation permeation and drug sensitivity. Here, we have used substituted-cysteine accessibility mutagenesis and thiol-modifying methanethiosulfonate (MTS) reagents to further probe the involvement of evolutionarily conserved sites within M9 (residues 342-363) and the adjacent exofacial re-entrant loop 5 between M9 and M10 (EL5; residues 364-415) of a cysteine-less variant of rat NHE1 on its kinetic and pharmacological properties. MTS treatment significantly reduced the activity of mutants containing substitutions within M9 (H353C, S355C, and G356C) and EL5 (G403C and S405C). In the absence of MTS, mutants S355C, G403C, and S405C showed modest to significant decreases in their apparent affinities for Na(+) o and/or H(+) i. In addition, mutations Y370C and E395C within EL5, whereas failing to confer sensitivity to MTS, nevertheless, reduced the affinity for Na(+) o, but not for H(+) i. The Y370C mutant also exhibited higher affinity for ethylisopropylamiloride, a competitive antagonist of Na(+) o transport. Collectively, these results further implicate helix M9 and EL5 of NHE1 as important elements involved in cation transport and inhibitor sensitivity, which may inform rational drug design.

Activation of AMP-activated protein kinase regulates hippocampal neuronal pH by recruiting Na(+)/H(+) exchanger NHE5 to the cell surface.

Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H(+)-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na(+)/H(+) exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress.