Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

A Ca2+ Release-activated Ca2+ (CRAC) Modulatory Domain (CMD) within STIM1 Mediates Fast Ca2+-dependent Inactivation of ORAI1 Channels

STIM1 and ORAI1, the two limiting components in the Ca²⁺ release-activated Ca²⁺ (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2–3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca²⁺, a decrease in intracellular Ca²⁺ chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba²⁺ substitution for extracellular Ca²⁺ completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca²⁺ entry by triggering fast Ca²⁺-dependent inactivation of ORAI/CRAC channels.The Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel is one of the best characterized store-operated entry pathways (1–7). Substantial efforts have led to identification of two key components of the CRAC channel machinery: the stromal interaction molecule 1 (STIM1), which is located in the endoplasmic reticulum and acts as a Ca²⁺ sensor (8–10), and ORAI1/CRACM1, the pore-forming subunit of the CRAC channel (11–13). Besides ORAI1, two further homologues named ORAI2 and ORAI3 belong to the ORAI channel family (12, 14).STIM1 senses endoplasmic reticulum store depletion primarily by its luminal EF-hand in its N terminus (8, 15), redistributes close to the plasma membrane, where it forms puncta-like structures, and co-clusters with ORAI1, leading to inward Ca²⁺ currents (12, 16–19). The STIM1 C terminus, located in the cytosol, contains two coiled-coil regions overlapping with an ezrin-radixin-moesin (ERM)-like domain followed by a serine/proline- and a lysine-rich region (2, 8, 20–22). Three recent studies have described the essential ORAI-activating region within the ERM domain, termed SOAR (Stim ORAI-activating region) (23), OASF (ORAI-activating small fragment) (24), and CAD (CRAC-activating domain) (25), including the second coiled coil domain and the following ∼55 amino acids. We and others have provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (26–28) that is mediated by a direct interaction of the STIM1 C terminus with ORAI1 C terminus probably involving the putative coiled-coil domain in the latter (27).Furthermore, different groups have proven that the C terminus of STIM1 is sufficient to activate CRAC as well as ORAI1 channels independent of store depletion (22–25, 27, 29). We have identified that OASF-(233–474) or shorter fragments exhibit further enhanced coupling to ORAI1 resulting in 3-fold increased constitutive Ca²⁺ currents. A STIM1 fragment containing an additional cluster of anionic amino acids C-terminal to position 474 displays weaker interaction with ORAI1 as well as reduced Ca²⁺ current comparable with that mediated by wild-type STIM1 C terminus. Hence, we have suggested that these 11 amino acids (474–485) act in a modulatory manner onto ORAI1; however, their detailed mechanistic impact within the STIM1/ORAI1 signaling machinery has remained so far unclear.In this study, we focused on the impact of this negative cluster on fast inactivation of STIM1-mediated ORAI Ca²⁺ currents. Lis et al. (30) have shown that all three ORAI homologues display distinct inactivation profiles, where ORAI2 and ORAI3 show a much more pronounced fast inactivation than ORAI1. Moreover, it has been reported (31) that different expression levels of STIM1 to ORAI1 affect the properties of CRAC current inactivation. Yamashita et al. (32) have demonstrated a linkage between the selectivity filter of ORAI1 and its Ca²⁺-dependent fast inactivation. Here we provide evidence that a cluster of acidic residues within the C terminus of STIM1 is involved in the fast inactivation of ORAI1 and further promotes that of ORAI3 and native CRAC currents.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

A Trk/HKT-Type K+ Transporter from Trypanosoma brucei

The molecular mechanisms of K⁺ homeostasis are only poorly understood for protozoan parasites. Trypanosoma brucei subsp. parasites, the causative agents of human sleeping sickness and nagana, are strictly extracellular and need to actively concentrate K⁺ from their hosts’ body fluids. The T. brucei genome contains two putative K⁺ channel genes, yet the trypanosomes are insensitive to K⁺ antagonists and K⁺ channel-blocking agents, and they do not spontaneously depolarize in response to high extracellular K⁺ concentrations. However, the trypanosomes are extremely sensitive to K⁺ ionophores such as valinomycin. Surprisingly, T. brucei possesses a member of the Trk/HKT superfamily of monovalent cation permeases which so far had only been known from bacteria, archaea, fungi, and plants. The protein was named TbHKT1 and functions as a Na⁺-independent K⁺ transporter when expressed in Escherichia coli, Saccharomyces cerevisiae, or Xenopus laevis oocytes. In trypanosomes, TbHKT1 is expressed in both the mammalian bloodstream stage and the Tsetse fly midgut stage; however, RNA interference (RNAi)-mediated silencing of TbHKT1 expression did not produce a growth phenotype in either stage. The presence of HKT genes in trypanosomatids adds a further piece to the enigmatic phylogeny of the Trk/HKT superfamily of K⁺ transporters. Parsimonial analysis suggests that the transporters were present in the first eukaryotes but subsequently lost in several of the major eukaryotic lineages, in at least four independent events.Potassium (K⁺) is the most abundant cation in the cytosol of any cell and hence an essential macronutrient for life on earth. Concentrative K⁺ uptake across the plasma membrane is energized directly by ATPases and indirectly by the negative membrane potential or by coupling, via symport or antiport, to other transport processes such as H⁺ flux. The ancestry of K⁺ transporters renders them ideal subjects for phylogenetic comparisons. Indeed, the different kinds of known K⁺ transporters—pumps, channels, permeases, symporters, and antiporters—are all found in bacteria (43). Eukaryotes do not appear to have invented further mechanisms of K⁺ transport; on the contrary, some families of K⁺ transporters were lost over the course of eukaryote evolution, particularly among the metazoa (53).The Trk/HKT superfamily (TC transporter classification 2.A.38 [43]) consists of bacterial TrkH and KtrB, plant HKT, and fungal Trk transporters (15). These proteins share a topology with 8 transmembrane (TM) domains and, sandwiched between odd- and even-numbered TM domains, 4 shorter hydrophobic helices that resemble the P-loops of K⁺ channels (14, 27, 55). In the K⁺ channel, these pore-forming loops end in the filter residues glycine-tyrosine-glycine, which coordinate K⁺ by means of their backbones’ carbonyl oxygens (13). The P-loop-like helices of Trk/HKT transporters end in a single conserved glycine (48), and these glycines have been shown to determine K⁺ selectivity over Na⁺ of the transporters (34, 49). Thus, a Trk/HKT monomer with 8 TM domains and 4 P-loops is thought to have a similar pore architecture to a K⁺ channel tetramer with two TM domains and one P-loop per subunit. The Trk/HKT transporters are important for cellular K⁺ acquisition in microorganisms, since trk null mutant yeast or bacteria exhibit growth phenotypes on media containing low K⁺ concentrations (20, 46). The roles of the Trk/HKT transporters in plants are more diverse, including Na⁺ distribution (10, 33, 47), osmoregulation (32), and salt tolerance (39). So far, no HKT/Trk transporter has been described from the metazoa or protista.Trypanosoma brucei subsp. parasites comprise the causative agents of human and livestock trypanosomosis: sleeping sickness and nagana, respectively. The distribution of the parasites is restricted by that of their vector, the blood-sucking tsetse fly (Glossina spp.), to the so-called tsetse belt comprising 36 countries between the Sahara desert and the Kalahari (3, 8). African trypanosomes proliferate extracellularly in the blood, evading the mammalian immune response by antigenic variation. Untreated sleeping sickness is fatal. There is an urgent need for new and better drugs since the current ones, the arsenical melarsoprol in particular, suffer from severe side effects (31). In the mammalian bloodstream, the parasites encounter a rich and steady supply of nutrients, readily imported by specific permeases or endocytosed via receptors (7). Research on trypanosomal nutrient uptake has so far concentrated on transporters of organic substrates: nucleobases, nucleosides, sugars, and amino acids (4, 12, 26, 30, 35, 56). Little is known about how the parasites import inorganic nutrients. The malaria parasite Plasmodium falciparum possesses two putative K⁺ channel subunits with 6 TM domains and one P-loop (19, 52). Disruption of an orthologous gene in Plasmodium berghei strongly impaired the development of the malaria parasites in the mosquito (18). However, these putative channels have not yet been proven to be permeable to K⁺. The T. brucei genome (6) is annotated to contain two putative K⁺ channels; in addition, a putative ATPase has been identified resembling fungal Na⁺/K⁺ efflux ATPases (5, 45). None of these has been addressed experimentally. Here we present the identification and characterization of TbHKT1 (Tb10.70.2940), a Trk/HKT-type K⁺ transporter from Trypanosoma brucei and representative of a new clade of Trk/HKT genes from kinetoplastid parasites.     

Heteromeric AtKC1��AKT1 Channels in Arabidopsis Roots Facilitate Growth under K+-limiting Conditions

Plant growth and development is driven by osmotic processes. Potassium represents the major osmotically active cation in plants cells. The uptake of this inorganic osmolyte from the soil in Arabidopsis involves a root K⁺ uptake module consisting of the two K⁺ channel α-subunits, AKT1 and AtKC1. AKT1-mediated potassium absorption from K⁺-depleted soil was shown to depend on the calcium-sensing proteins CBL1/9 and their interacting kinase CIPK23. Here we show that upon activation by the CBL·CIPK complex in low external potassium homomeric AKT1 channels open at voltages positive of E_K, a condition resulting in cellular K⁺ leakage. Although at submillimolar external potassium an intrinsic K⁺ sensor reduces AKT1 channel cord conductance, loss of cytosolic potassium is not completely abolished under these conditions. Depending on channel activity and the actual potassium gradients, this channel-mediated K⁺ loss results in impaired plant growth in the atkc1 mutant. Incorporation of the AtKC1 subunit into the channel complex, however, modulates the properties of the K⁺ uptake module to prevent K⁺ loss. Upon assembly of AKT1 and AtKC1, the activation threshold of the root inward rectifier voltage gate is shifted negative by approximately −70 mV. Additionally, the channel conductance gains a hypersensitive K⁺ dependence. Together, these two processes appear to represent a safety strategy preventing K⁺ loss through the uptake channels under physiological conditions. Similar growth retardation phenotypes of akt1 and atkc1 loss-of-function mutants in response to limiting K⁺ supply further support such functional interdependence of AKT1 and AtKC1. Taken together, these findings suggest an essential role of AtKC1-like subunits for root K⁺ uptake and K⁺ homeostasis when plants experience conditions of K⁺ limitation.Fundamental plant functions such as control of the membrane potential, osmo-regulation, and turgor-driven growth and movements are based on the availability to gain high cellular potassium concentrations (1). The absorption of this inorganic osmolyte from the soil by the root therefore represents a pivotal process for plant life. Classical experiments by Epstein et al. in 1963 (2) described K⁺ root uptake as a biphasic process mediated by two uptake mechanisms: high affinity potassium transport with apparent affinities of ∼20 μm and a low affinity transport system with K_m values in the millimolar range. During the last decades several molecular components of potassium transport systems have been identified and functionally characterized in plants (3, 4). Mutant analyses, heterologous expression, as well as radiotracer uptake experiments characterized the K⁺ channels AKT1·AtKC1 and members of the HAK·KT·KUP family as major components of the Arabidopsis thaliana root-localized potassium transport system (5–9). In this study we focused on AKT1 and AtKC1, members of the Arabidopsis Shaker-like K⁺ channel family. AKT1 is a voltage-dependent inward-rectifying K⁺ channel mediating potassium uptake over a wide range of external potassium concentrations (10–15). Root cells of the akt1-1 loss-of-function mutant completely lack inward rectifying K⁺ currents (12). As a consequence the growth of akt1-1 seedlings is strongly impaired on low potassium medium (100 μm and less) (11, 12, 15). Rescue of yeast growth on 20 μm K⁺ and patch clamp experiments (16, 17) directly demonstrated that plant inward rectifying K⁺ channels are capable of serving as high affinity potassium uptake transporters. AtKC1 shares its expression pattern with AKT1 (18–20). AtKC1 α-subunits, however, neither form functional channels in akt1-1 knock-out plants nor in heterologous expression systems. In contrast to root cells of akt1-1 loss of function mutants, root protoplasts of AtKC1 null mutants (atkc1-f) still exhibit inward rectifying potassium currents most likely derived from homomeric AKT1 tetramers (20). Inward K⁺ currents in this atkc1-f mutant were characterized by a more positive activation voltage. These data suggested that the AtKC1 α-subunits do not form K⁺ channels per se but modulate the properties of the AKT1·AtKC1 heterocomplex (20–22). Previously, two groups in their ground-breaking studies demonstrated that AKT1 is activated by the CBL²-interacting, serine/threonine kinase, CIPK23, particularly under low K⁺ conditions (23, 24). CIPK23 itself was shown to be activated by the two calcineurin B-like proteins, CBL1 and 9, acting in a Ca²⁺-dependent manner upstream of CIPK23 (25, 26). Genetic disruption of these elements resulted in transgenic plants exhibiting a phenotype comparable with that of the AKT1 loss of function mutant. This regulatory system, based on a calcium sensor, a protein kinase, and a K⁺ channel, was functionally reconstituted in Xenopus oocytes (23, 24, 27), suggesting that these elements are essential and sufficient to operate as a low K⁺-sensitive potassium uptake system. Here we report on the physiological properties of the heteromeric K⁺ uptake module formed by the predominant root potassium uptake channel subunits, AKT1 and AtKC1 and its regulating kinase complex, CBL1 and CIPK23. Our studies show that the physical interaction of the CBL1·CIPK23 complex is specific for AKT1 channels and does not involve the AtKC1 subunit. AKT1 possesses a K⁺ (absence) sensor affecting channel activity at submillimolar K⁺ concentrations by strongly reducing its maximal cord conductance. Despite this K⁺ sensor, upon activation, AKT1 homomeric channels were shown to represent a potassium leak at low external potassium concentrations. Integration of AtKC1 into the K⁺ uptake module, however, prevented potassium loss by modulating both the voltage sensor and conductance in the channel complex. Moreover, activation of the AKT1-like maize channel ZMK1 by CBL1·CIPK23 suggests a conserved interaction and regulation across monocot and dicotyledonous plant species. Our biophysical studies as well as growth assays with plant mutant lines lacking the respective channels underline that acquisition of potassium under limiting K⁺ conditions is mediated via the root AKT1·AtKC1 K⁺ uptake channel complex.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Mutation of Asparagine 76 in the Center of Glutamine Transporter SNAT3 Modulates Substrate-induced Conductances and Na+ Binding

The glutamine transporter SLC38A3 (SNAT3) plays an important role in the release of glutamine from brain astrocytes and the uptake of glutamine into hepatocytes. It is related to the vesicular GABA (γ-aminobutyric acid) transporter and the SLC36 family of proton-amino acid cotransporters. The transporter carries out electroneutral Na⁺-glutamine cotransport-H⁺ antiport. In addition, substrate-induced uncoupled cation currents are observed. Mutation of asparagine 76 to glutamine or histidine in predicted transmembrane helix 1 abolished all substrate-induced currents. Mutation of asparagine 76 to aspartate rendered the transporter Na⁺-independent and resulted in a gain of a large substrate-induced chloride conductance in the absence of Na⁺. Thus, a single residue is critical for coupled and uncoupled ion flows in the glutamine transporter SNAT3. Homology modeling of SNAT3 along the structure of the related benzyl-hydantoin permease from Microbacterium liquefaciens reveals that Asn-76 is likely to be located in the center of the membrane close to the translocation pore and forms part of the predicted Na⁺ -binding site.The amino acid and auxin permease superfamily comprises a wide variety of transport proteins. In mammals, three distinct solute carrier families (SLC) belong to this superfamily, namely SLC32, SLC36, and SLC38 (1). Despite belonging to the same superfamily, the three solute carrier families have different transport mechanisms. The SLC32 family has only one member, the vesicular inhibitory amino acid transporter, which supposedly carries out a H⁺-GABA (γ-aminobutyric acid) antiport (2). The SLC36 family comprises four members, two of which have been characterized in more detail. These are the proton amino acid cotransporters 1 and 2 (PAT1 and 2) that carry out glycine and proline uptake in kidney and intestine and are mutated in iminoglycinuria (3, 4). The SLC38 family is comprised of 11 members, 5 of which have been characterized in more detail (5). Two different transport mechanisms are found within this family, namely the Na⁺-amino acid cotransporters SNAT1, SNAT2, and SNAT4 and the Na⁺-amino acid cotransporters-H⁺-antiporters SNAT3 and SNAT5. Transporters of the superfamily play a key role in inhibitory and excitatory neurotransmission, metabolite absorption, and liver metabolism. Despite their important roles in mammalian physiology, relatively little is known about the structure and function of these transporters.The activity of ion-coupled membrane transporters is frequently associated with currents which de- or hyperpolarize the cell membrane. These currents may be due to electrogenic transport stoichiometry and/or to a non-stoichiometric ion conductance (6). Transport-associated ion conductances have been identified in a number of transporters but have been particularly well studied in several Na⁺-coupled neurotransmitter transporters (7–11). Transport-associated conductances have also been observed in electroneutral transporters that do not carry out net charge movement (8, 12–15). The glutamine transporter SNAT3, for instance, has a transport mechanism in which glutamine uptake is coupled to the cotransport of 1Na⁺ and the antiport of 1H⁺ and, hence, is unaffected by changes of the membrane potential (13, 16). Despite the electroneutral transport mechanism, substrate uptake is accompanied by inward currents, which are carried by cations below pH 7 and by protons at alkaline pH. In addition, a substrate-independent cation conductance and a Na⁺/H⁺ exchange activity has been observed (17). Non-stoichiometric currents can be mediated by the same ions that are involved in the coupled transport process, such as in the case of SNAT3, but may also be carried by different ions. Stoichiometric glutamate transport, for instance, involves Na⁺, H⁺, and K⁺ ions, whereas the glutamate transport-associated conductance is carried by chloride (18).A crucial question concerning transporter-associated ion conductances is whether the conducting pore coincides with the translocation pathway of the substrate and whether both use the same critical residues. In the case of the glutamate transporters, evidence has been presented suggesting that different residues are critical for the anion conductance than for substrate transport (19, 20) but that they all line the same pathway (21). Here we show that asparagine 76 of SNAT3 is critical for substrate-induced ion conductance and affects binding of the cosubstrate Na⁺. In addition we show that this residue is likely to be localized in the translocation pore in the center of the membrane.     

Paradoxical Condensation of Copper with Elevated ��-Amyloid in Lipid Rafts under Cellular Copper Deficiency Conditions: IMPLICATIONS FOR ALZHEIMER DISEASE*

Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), β-amyloid peptide (Aβ) aggregation, and amyloid formation. Aβ·copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the β-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. β- and γ-secretases, and Aβ have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Aβ synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Aβ·copper complexes are more likely to form. This explains the paradoxical hypermetallation of Aβ with copper under tissue copper deficiency conditions in AD.Imbalance of metal ions has been recognized as one of the key factors in the pathogenesis of Alzheimer disease (AD).² Aberrant interactions between copper or zinc with the β-amyloid peptide (Aβ) released into the glutamatergic synaptic cleft vicinity could result in the formation of toxic Aβ oligomers and aggregation into plaques characteristic of AD brains (reviewed in Ref. 1). Copper, iron, and zinc are highly concentrated in extracellular plaques (2, 3), and yet brain tissues from AD (4–6) and human β-amyloid precursor protein (APP) transgenic mice (7–10) are paradoxically copper deficient compared with age-matched controls. Elevation of intracellular copper levels by genetic, dietary, and pharmacological manipulations in both AD transgenic animal and cell culture models is able to attenuate Aβ production (7, 9, 11–15). However, the underlying mechanism is at present unclear.Abnormal cholesterol metabolism is also a contributing factor in the pathogenesis of AD. Hypercholesterolemia increases the risk of developing AD-like pathology in a transgenic mouse model (16). Epidemiological and animal model studies show that a hypercholesterolemic diet is associated with Aβ accumulation and accelerated cognitive decline, both of which are further aggravated by high dietary copper (17, 18). In contrast, biochemical depletion of cholesterol using statins, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, and methyl-β-cyclodextrin, a cholesterol sequestering agent, inhibit Aβ production in animal and cell culture models (19–25).Cholesterol is enriched in lipid rafts, membrane microdomains implicated in Aβ generation from APP cleavage by β- and γ-secretases. Recruitment of BACE1 (β-secretase) into lipid rafts increases the production of sAPP_β and Aβ (23, 26). The β-secretase-cleaved APP C-terminal fragment (β-CTF), and γ-secretase, a multiprotein complex composed of presenilin (PS1 or PS2), nicastrin (Nct), PEN-2 and APH-1, colocalize to lipid rafts (27). The accumulation of Aβ in lipid rafts isolated from AD and APP transgenic mice brains (28) provided further evidence that cholesterol plays a role in APP processing and Aβ generation.Currently, copper and cholesterol have been reported to modulate APP processing independently. However, evidence indicates that, despite tissue copper deficiency, Aβ·Cu²⁺ complexes form in AD that catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides (e.g. hydroxynonenal and malondialdehyde), which contribute to oxidative damage observed in AD (29–35). The underlying mechanism leading to the formation of pathological Aβ·Cu²⁺ complexes is unknown. In this study, we show that copper alters the structure of lipid rafts, and attenuates Aβ synthesis in lipid rafts by inhibition of APP endocytosis. We also identify a paradoxical inverse relationship between total cellular copper levels and copper distribution to lipid rafts, which appear to possess a privileged pool of copper where Aβ is more likely to interact with Cu²⁺ under copper-deficiency conditions to form Aβ·Cu²⁺ complexes. These data provide a novel mechanism by which cellular copper deficiency in AD could foster an environment for potentially adverse interactions between Aβ, copper, and cholesterol in lipid rafts.     

Inflammasome-dependent Caspase-1 Activation in Cervical Epithelial Cells Stimulates Growth of the Intracellular Pathogen Chlamydia trachomatis

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K⁺ through glibenclamide-sensitive K⁺ channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive oxygen species are responsible for NLRP3-dependent caspase-1 activation in the infected cells. In monocytes and macrophages, caspase-1 is involved in processing and secretion of pro-inflammatory cytokines such as interleukin-1β. However, in epithelial cells, which are not known to secrete large quantities of interleukin-1β, caspase-1 has been shown previously to enhance lipid metabolism. Here we show that, in cervical epithelial cells, caspase-1 activation is required for optimal growth of the intracellular chlamydiae.Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States, and it is the leading cause of preventable blindness in the world (1–5). Untreated, C. trachomatis infection in women can cause pelvic inflammatory disease, which can lead to infertility and ectopic pregnancy because of scarring of the ovaries and the Fallopian tubes (6). Infection by the lymphogranuloma venereum (LGV)² strain of C. trachomatis, which has become more common in North America and Europe (7, 8), is characterized by swelling and inflammation of the lymph nodes in the groin (9).Chlamydiae are intracellular pathogens that preferentially infect epithelial mucosa and have a biphasic infection cycle (10). A metabolically inactive form, the elementary body, infects the epithelial host cells through entry vesicles that avoid fusion with host cell lysosomes and develop into a membrane-bound inclusion (11–13). Despite their intravacuolar localization, chlamydiae are still able to acquire nutrients from the host cell and interact with host-cell signaling pathways (13–23). Within a few hours, the elementary bodies differentiate into larger, metabolically active reticulate bodies, which proliferate but are noninfectious. Depending on the strain of C. trachomatis, the reticulate bodies transform back into elementary bodies after 1–3 days and are released into the extracellular medium to infect other cells (11, 24, 25). Chlamydial species possess a type III secretion (T3S) system that secretes bacterial virulence factors into host cell cytosol and may control interactions between the inclusion and host-cell compartments (26).Long before the adaptive immune response is activated, infected epithelial cells produce proinflammatory cytokines and chemokines, including interleukin (IL)-6, IL-8, and granulocyte-macrophage colony-stimulating factor (27), which recruit neutrophils to the site of infection and activate other immune effector cells. However, in many cases the immune system fails to clear the infection, and the chronic release of cytokines becomes a major contributor to the scarring and damage associated with the infection (28–30).The innate immune response during C. trachomatis infection is initiated by chlamydial pathogen-associated molecular patterns, including lipopolysaccharides, which bind to pattern recognition receptors such as Toll-like receptors and cytosolic NOD-like receptors (NLRs), ultimately promoting pro-inflammatory cytokine gene expression and secretion of the cytokine proteins (31–37). However, secretion of the key pro-inflammatory cytokine IL-1β is tightly regulated (38). First, pro-IL-1β is produced following activation of pattern recognition receptor, and the precursor is then cleaved into the mature form by the pro-inflammatory cysteine protease, caspase-1 (also known as interleukin-1 converting enzyme or ICE). The mechanism by which caspase-1 is activated in response to infection or tissue damage was found to be modulated by a macromolecular protein complex termed the “inflammasome,” which consists of an NLR family member, an adaptor protein (apoptosis-associated speck-like protein containing a caspase activation recruitment domain or ASC), and an inactive caspase-1 precursor (pro-caspase-1) (39, 40). Previous studies demonstrated that IL-1β is produced in response to chlamydial infection in dendritic cells, macrophages, and monocytes (41–44). Moreover, C. trachomatis or Chlamydia caviae infection activates caspase-1 in epithelial cells or monocytes (43, 45, 46). However, whether caspase-1 activation during chlamydial infection requires the formation of an inflammasome remains unclear.Previous studies have shown that different pathogens can cause inflammasome-mediated caspase-1 activation in macrophages and monocytes (47). However, epithelial cells lining mucosal surfaces are not only the preferred target for chlamydial infection and other intracellular pathogens but also play an important role in early host immune response to infection by secreting proinflammatory cytokines and chemokines (27). Although epithelial cells are not known to secrete large amounts of IL-1β, inflammasome-dependent caspase-1 activation in epithelial cells is known to contribute to lipid metabolism and membrane regeneration in epithelial cells damaged by the membrane-disrupting toxin, aerolysin (48). As lipids are sorted from the Golgi apparatus to the chlamydial inclusion (13, 15, 49), we therefore investigated whether C. trachomatis induces caspase-1 activation in epithelial cells via the assembly of an inflammasome. We demonstrated that C. trachomatis-induced caspase-1 activation is mediated by an inflammasome containing the NLR member, NLRP3. Several studies have demonstrated the involvement of T3S apparatus in inflammasome-mediated caspase-1 activation by different pathogens in macrophages and monocytes (50–56). Therefore, we further investigated the mechanism by which C. trachomatis triggers the formation of the NLRP3 inflammasome. Our results showed that metabolically active chlamydiae, relying on their T3S apparatus, cause K⁺ efflux, which in turn leads to formation of reactive oxygen species (ROS) and ultimately NLRP3-dependent caspase-1 activation. Epithelial cells do not typically secrete large amounts of IL-1β; instead, caspase-1 activation in cervical epithelial cells contributes to development of the chlamydial inclusion.     

Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport

Human concentrative nucleoside transporter, hCNT3, mediates Na⁺/nucleoside and H⁺/nucleoside co-transport. We describe a new approach to monitor H⁺/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosolic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H⁺-coupled nucleoside transport, pH 5.5, under both Na⁺-free and Na⁺-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mm uridine, providing the first direct evidence for H⁺-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H⁺ flux) in the presence or absence of Na⁺ (1.09 ± 0.11 or 1.18 ± 0.32 mm min⁻¹, respectively). This suggests that in acidic Na⁺-containing conditions, 1 Na⁺ and 1 H⁺ are transported per uridine molecule, while in acidic Na⁺-free conditions, 1 H⁺ alone is transported/uridine. In acid environments, including renal proximal tubule, H⁺/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H⁺-coupled transport proteins.Nucleosides are hydrophilic molecules that require transport proteins to mediate their movement across the plasma membrane (1). Human (h)⁷ nucleoside transport (NT) proteins catalyze the vectorial transport of nucleosides, using either concentrative (C) or equilibrative (E) mechanisms (2). hCNTs use either a Na⁺ or H⁺ gradient to accumulate nucleosides against their concentration gradient, whereas hENTs mediate facilitated diffusion of nucleosides down their concentration gradient (3). Nucleoside transporters also transport anti-cancer and anti-viral drugs, and cellular expression of nucleoside transporters is important in cancer therapy as well as in the treatment of cardiovascular, parasitic, and viral diseases (4, 5).Members of the SLC28 family of concentrative nucleoside transporters (CNTs) divide into two phylogenetic subfamilies: hCNT1/2 belonging to one subfamily, and hCNT3 to the other (6–8). Cation substitution and charge/flux ratio studies suggest that hCNT1/2 couple the inward movement of nucleoside to the Na⁺ electrochemical gradient with a 1:1 stoichiometry, whereas hCNT3 can couple nucleoside transport to either the Na⁺ gradient (2 Na⁺:1 nucleoside) or a H⁺ gradient (1 H⁺:1 nucleoside) in the absence of Na⁺ (9, 10). The 2:1 coupling ratio of hCNT3 allows it to develop a trans-membrane nucleoside concentration gradient up to 10-fold higher than that of hCNT1 or hCNT2 (9, 11). At pH 5.5, hCNT3 also transports uridine in the presence of Na⁺ with a 2 cation:1 nucleoside stoichiometry, which raises the possibility that 1 H⁺ and 1 Na⁺ may be transported per nucleoside molecule in these conditions (9–12). Up to this point, however, there has been no direct demonstration that hCNT3 can transport H⁺.Concentrative nucleoside transport has previously been investigated using the Xenopus laevis oocyte expression system and both electrophysiology (two-microelectrode voltage clamp technique) and radioisotope flux measurements (6–9, 12). Electrophysiological experiments are advantageous in that they measure the current induced by addition of substrate in real-time, but they are time-consuming and require specialized equipment and skills. Radioisotope flux assays measure the accumulation of radiolabeled substrate. The need for radiolabeled substrate restricts the range of permeants able to be studied. In addition, radioisotope flux assays are not done in real-time and are labor-intensive, requiring large numbers of oocytes.An attractive alternative approach for the study of hCNT3 would be to measure pH in the immediate vicinity of its intracellular face during H⁺/nucleoside co-transport. These measurements could take advantage of the remarkable progress achieved in the development of genetically encoded fluorophores (13). Indeed, all members of the extended family of homologues and variants of the Aequorea victoria green fluorescent protein (avGFP) exhibit pH-dependent changes in their fluorescent intensity. The spectral changes that occur upon a change in pH can be intensiometric (14), excitation ratiometric (14), emission ratiometric (15), or both excitation and emission ratiometric (16). The apparent pK_a (pK_a′, equal to the pH at which the fluorescence is half-maximal in intensity) for a specific fluorescent protein (FP) is acutely dependent on specific amino acid substitutions in close proximity to the chromophore and can range from less than 3 (17, 18) to greater than 8 (19). Variants with pK_a′ values that are relatively close to intracellular pH values (i.e. ∼7.3 for the mammalian cytosol (20)) are particularly useful as genetically encoded biosensors for dynamic measurement of proton concentrations in living cells.A major development in the area of FP technology has been the identification (21) and subsequent optimization (22, 23) of red fluorescent protein (RFP) homologues of avGFP. The first (monomeric RFP 1 (mRFP1)) (23) and second (the mFruit series) (22) generation-optimized RFPs, derived from tetrameric Discosoma RFP (21), suffer from relatively low brightness relative to other common hues of FP. For example, of the three most red-shifted second generation mFruit variants (mTangerine, mStrawberry, and mCherry) (22), the brightest (mStrawberry) has only 44% of the intrinsic brightness (proportional to the product of extinction coefficient (ϵ) and quantum yield (Φ)) of the popular yellow FP (YFP) Citrine (24) and 76% of the brightness of enhanced avGFP. This limitation has been partially addressed by third generation mRFPs, specifically mApple and TagRFP-T, with fluorescent brightness values on par with, or better than, that of enhanced avGFP (25).Generally speaking, the most red-shifted RFPs derived from Discosoma RFP are relatively pH-insensitive, with the majority of variants having pK_a′ values < 5 (22, 25). A notable exception is the recently reported mApple variant with a pK_a′ of 6.5 (25). The more blue-shifted of the mFruit variants (i.e. mOrange) also have pK_a′ values of 6.5 (22). Several variants of mRFP1 with pK_a′ values >7.5 have been previously reported (26).Here we report the engineering of a pH-sensitive mFruit variant through multiple rounds of directed evolution by random mutagenesis. This RFP, called mNectarine, is appropriate to measure physiological pH changes in mammalian cells, because it has a pK_a′ of 6.9. We have developed a new method to measure H⁺/nucleoside co-transport in mammalian cells, which utilizes hCNT3''s H⁺ coupling characteristics and the pH sensitivity of mNectarine. We fused mNectarine to the cytosolic N terminus of hCNT3 to generate mNect.hCNT3. Fusion of the fluorescent H⁺ sensor to hCNT3 enabled measurement of pH at the intracellular surface of hCNT3, and provided insight into the mechanism of hCNT3 H⁺/uridine co-transport.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Conformational Properties of ��-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP^C) to a disease-specific isomer (PrP^Sc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP^Sc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP^C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrP^C structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)² (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrP^Sc, differs from the normal cellular form (PrP^C) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrP^C, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrP^C to PrP^Sc. The most coherent and general model proposed thus far is that PrP^C fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrP^Sc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrP^Sc-dependent conversion reactions (18–20) or through conversion of PrP^C in the absence of a PrP^Sc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrP^Sc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrP^C and PrP^Sc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrP^Sc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrP^Sc (but not PrP^C) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrP^Sc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP.     

Aqueous Accessibility to the Transmembrane Regions of Subunit c of the Escherichia coli F1F0 ATP Synthase

Rotary catalysis in F₁F₀ ATP synthase is powered by proton translocation through the membrane-embedded F₀ sector. Proton binding and release occur in the middle of the membrane at Asp-61 on transmembrane helix (TMH) 2 of subunit c. Previously the reactivity of Cys substituted into TMH2 revealed extensive aqueous access at the cytoplasmic side as probed with Ag⁺ and other thiolate-directed reagents. The analysis of aqueous accessibility of membrane-embedded regions in subunit c was extended here to TMH1 and the periplasmic side of TMH2. The Ag⁺ sensitivity of Cys substitutions was more limited on the periplasmic versus cytoplasmic side of TMH2. In TMH1, Ag⁺ sensitivity was restricted to a pocket of four residues lying directly behind Asp-61. Aqueous accessibility was also probed using Cd²⁺, a membrane-impermeant soft metal ion with properties similar to Ag⁺. Cd²⁺ inhibition was restricted to the I28C substitution in TMH1 and residues surrounding Asp-61 in TMH2. The overall pattern of inhibition, by all of the reagents tested, indicates highest accessibility on the cytoplasmic side of TMH2 and in a pocket of residues around Asp-61, including proximal residues in TMH1. Additionally subunit a was shown to mediate access to this region by the membrane-impermeant probe 2-(trimethylammonium)ethyl methanethiosulfonate. Based upon these results and other information, a pocket of aqueous accessible residues, bordered by the peripheral surface of TMH4 of subunit a, is proposed to extend from the cytoplasmic side of cTMH2 to Asp-61 in the center of the membrane.F₁F₀ ATP synthase utilizes the energy stored in an H⁺ or Na⁺ electrochemical gradient to synthesize ATP in bacteria, mitochondria, and chloroplasts (1–4). The ATP synthase complex is composed of two sectors, i.e. a water-soluble F₁ sector that is bound to a membrane-embedded F₀ sector. In bacteria, F₁ is composed of five subunits in an α₃β₃γδϵ ratio and contains three catalytic sites for ATP synthesis and/or hydrolysis centered at the α-β subunit interfaces. F₀ is composed of three subunits in an a₁b₂c_10–15 ratio and functions as the ion-conducting pathway (5–9). Ion translocation through F₀ drives rotation of a cylindrical ring of c-subunits that is coupled to rotation of the γ subunit within the (αβ)₃ hexamer of F₁ to force conformational changes in the three active sites and in turn drive synthesis of ATP by the binding change mechanism (1–4, 10–13).Subunit c of F₀ folds in the membrane as a hairpin of two extended α-helices. In Escherichia coli, 10 copies of subunit c pack together to form a decameric ring with TMH1² on the inside and TMH2 on the periphery (6, 14). An atomic resolution structure of the Na⁺-translocating c₁₁-ring from Ilyobacter tartaricus was recently published by Meier et al. (8). In the c₁₁ structure, the Na⁺ binding site is formed by two interacting c subunits. The essential Na⁺-binding Glu residue, which corresponds to Asp-61 in E. coli, is located in TMH2 at the middle of the lipid bilayer. Subunit a consists of five transmembrane helices, four of which likely interact as a four-helix bundle (15–18). Subunit a lies on the periphery of the c-ring with TMHs 4 and 5 from subunit a and TMH2 from subunit c forming the a-c interface (18–21). During ion translocation through F₀, the essential Arg-210 on TMH4 of subunit a is postulated to facilitate the protonation/deprotonation cycle at Asp-61 of subunit c and cause the rotation of the c-ring past the stationary subunit a (3, 4, 19).Chemical modification of cysteine-substituted transmembrane proteins has been widely used as a means of probing the aqueous accessible regions (22–24). The reactivity of a substituted cysteine to thiolate-directed probes provides an indication of aqueous accessibility because the reactive thiolate species is preferentially formed in an aqueous environment. The aqueous accessibility of the five TMHs in subunit a of E. coli F₀ has been probed using Ag⁺ and NEM (19, 25–27). The results suggest the presence of an aqueous accessible channel in subunit a in the center of TMHs 2–5 extending from the periplasm to the center of the membrane. Protons entering through this periplasmic access channel are postulated to bind to the essential Asp-61 residues of the c-ring and exit to the cytoplasm by a still uncertain pathway at the peripheral face of aTMH4 with protonation/deprotonation of Asp-61 driving c-ring rotation.During H⁺-driven ATP synthesis, two models for the pathway by which H⁺ or Na⁺ exit to the cytoplasm have been proposed. The first model proposes that the ions bound at Asp-61 exit to the cytoplasm via a half-channel composed at least partially by residues in TMH4 of subunit a (25–27). Chemical modification studies of Cys-substituted subunit a of E. coli revealed an aqueous accessible surface of TMH4 that includes the essential Arg-210 residue, which extended from the center of the membrane to the cytoplasm, suggesting that the ion exit channel may lie at the a-c interface (19, 25). Alternatively studies of the c-ring from the I. tartaricus enzyme indicate that Na⁺ can access Glu-65 in the absence of other F₀ subunits, suggesting an intrinsic channel in subunit c (28, 29). However, no such channel was apparent in the crystal structure of the c₁₁-ring (8). In a previous study (30), we probed the thiolate reactivity of Cys substitutions in the cytoplasmic half of TMH2 in subunit c. These experiments revealed extensive reactivity to sulfhydryl-directed reagents on the peripheral face of cTMH2, supporting the presence of the cytoplasmic exit channel at the a-c interface. In this study, we extended the survey of aqueous accessibility in transmembrane regions by probing thiolate reactivity of Cys substitutions in TMH1 and in the periplasmic half of TMH2. The reactivity of Cys substituted into these regions proved to be more limited. Only a small region of TMH1, lying directly behind Asp-61, was reactive with Ag⁺. In addition to Ag⁺, we used Cd²⁺ as a complementary, membrane-impermeant probe for aqueous accessibility. The survey of Cd²⁺ sensitivity confirmed that aqueous accessibility from the cytoplasm is much greater for residues packing at the periphery of the c-ring. The experiments reported here distinguish the aqueous accessible and inaccessible regions of the c-ring and strengthen evidence that the cytoplasmic H⁺ exit channel is situated at the a-c interface.     

Substituted Cysteine Accessibility Method Analysis of Human Concentrative Nucleoside Transporter hCNT3 Reveals a Novel Discontinuous Region of Functional Importance within the CNT Family Motif (G/A)XKX3NEFVA(Y/M/F)

The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na⁺-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na⁺ and/or H⁺ electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C−, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11–13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX₃NEFVA(Y/M/F).Specialized nucleoside transporter proteins are required for passage of nucleosides and hydrophilic nucleoside analogs across biological membranes. Physiologically, nucleosides serve as nucleotide precursors in salvage pathways, and pharmacologically nucleoside analogs are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases (1, 2). Additionally, adenosine modulates numerous cellular events via purino-receptor cell signaling pathways, including neurotransmission, vascular tone, immune responses, and other physiological processes (3, 4).Human nucleoside transporter proteins are divided into two families: the SLC29 ENT (equilibrative nucleoside transporter) family and the SLC28 CNT (concentrative nucleoside transporter) family (3, 5–7). hENTs³ mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types (8). Additionally, the hENT2 isoform is capable of nucleobase transport (9). hCNTs, in contrast, are inwardly directed Na⁺-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types (10, 11). hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, and couple Na⁺/nucleoside cotransport with 1:1 stoichiometry (12–18). In contrast, hCNT3 is broadly selective for both pyrimidine and purine nucleosides and couples Na⁺/nucleoside cotransport with 2:1 stoichiometry (10, 18, 19). hCNT3 is also capable of H⁺/nucleoside cotransport with a coupling stoichiometry of 1:1, whereby one of the two Na⁺ binding sites also functionally interacts with H⁺ (18, 19).Current models of CNT topology have 13 putative transmembrane helices (TMs) (10, 14, 16, 20). Two additional TMs (designated 5A and 11A) are weakly predicted by computer algorithms (20), and immunocytochemical experiments with site-specific antibodies and studies of native and introduced glycosylation sites have confirmed an intracellular N terminus and an extracellular C terminus (20). Chimeric studies involving hCNTs and hfCNT, a CNT from the ancient marine prevertebrate, the Pacific hagfish Eptatretus stouti, have revealed that the functional domains responsible for CNT nucleoside selectivity and cation coupling reside within the C-terminal TM 7–13 half of the protein (19, 21). NupC, an H⁺-coupled CNT family member from Escherichia coli, lacks TMs 1–3 but otherwise shares a topology similar to that of its eukaryote counterparts (22, 23).A functional cysteineless version of hCNT3 has been generated by mutagenesis of endogenous cysteine residues to serine, resulting in the cysteineless construct hCNT3C− employed originally in a yeast expression system for substituted cysteine accessibility method (SCAM) analysis of TMs 11, 12, and 13 using methanethiosulfonate (MTS) reagents (24). Subsequently, we have also characterized hCNT3C− in the Xenopus oocyte expression system (25) and have initiated SCAM analyses with the alternative thiol-specific reagent p-chloromercuribenzene sulfonate (PCMBS) (26). Measured by transport inhibition, reactivity of introduced cysteine residues with PCMBS, which is both membrane-impermeant and hydrophilic, indicates pore-lining status and access from the extracellular medium; the ability of a permeant to protect against this inhibition denotes location within, or closely adjacent to, the permeant-binding pocket (27, 28). Continuing the investigation of hCNT3 C-terminal membrane topology and function, the present study reports results of PCMBS SCAM analyses of TMs 11–13, including loop regions linking the putative TMs not previously studied using MTS reagents.In earlier structure/function studies of hCNT3, we identified a cluster of conformationally sensitive residue positions in TM 12 (Ile⁵⁵⁴, Tyr⁵⁵⁸, and Cys⁵⁶¹) that exhibit H⁺-activated inhibition by PCMBS, with uridine protection evident for Tyr⁵⁵⁸ and Cys⁵⁶¹ (26). Located deeper within the plane of the membrane, other uridine-protectable residue positions in TM 12 were PCMBS-sensitive in both H⁺- and Na⁺-containing media (26). hCNT3 Glu⁵¹⁹ and the corresponding residue in hCNT1 (Glu⁴⁹⁸) in region TM 11A were also identified as having key roles in permeant and cation binding and translocation (29, 30), and hCNT3 E519C showed inhibition of uridine uptake by PCMBS (30). Centrally positioned within the highly conserved CNT family motif (G/A)XKX₃NEFVA(Y/M/F), residue 519 is proposed to be a direct participant in cation coupling via the common hCNT3 Na⁺/H⁺-binding site that, in other CNTs, is either Na⁺-specific (e.g. hCNT1) or H⁺-specific (e.g. NupC) (30).Building upon the prior work with MTS reagents and other structure/function studies of hCNT3, the present study identified new residues of functional importance in the C-terminal one-third of hCNT3, established the orientations and α-helical structures of TMs 11–13, and determined a novel membrane-associated topology for the TM 11A region of the protein. A revised CNT membrane architecture is proposed.     

LFA-1 Regulates CD8+ T Cell Activation via T Cell Receptor-mediated and LFA-1-mediated Erk1/2 Signal Pathways

LFA-1 regulates T cell activation and signal transduction through the immunological synapse. T cell receptor (TCR) stimulation rapidly activates LFA-1, which provides unique LFA-1-dependent signals to promote T cell activation. However, the detailed molecular pathways that regulate these processes and the precise mechanism by which LFA-1 contributes to TCR activation remain unclear. We found LFA-1 directly participates in Erk1/2 signaling upon TCR stimulation in CD8⁺ T cells. The presence of LFA-1, not ligand binding, is required for the TCR-mediated Erk1/2 signal pathway. LFA-1-deficient T cells have defects in sustained Erk1/2 signaling and TCR/CD3 clustering, which subsequently prevents MTOC reorientation, cell cycle progression, and mitosis. LFA-1 regulates the TCR-mediated Erk1/2 signal pathway in the context of immunological synapse for recruitment and amplification of the Erk1/2 signal. In addition, LFA-1 ligation with ICAM-1 generates an additional Erk1/2 signal, which synergizes with the existing TCR-mediated Erk1/2 signal to enhance T cell activation. Thus, LFA-1 contributes to CD8⁺ T cell activation through two distinct signal pathways. We demonstrated that the function of LFA-1 is to enhance TCR signaling through the immunological synapse and deliver distinct signals in CD8⁺ T cell activation.Leukocyte function-associated antigen-1 (LFA-1)² plays an important role in regulating leukocyte adhesion and T cell activation (1, 2). LFA-1 consists of the αL (CD11a) and β2 (CD18) subunits. The ligands for LFA-1 include intercellular adhesion molecular-1 (ICAM-1), ICAM-2, and ICAM-3 (3). LFA-1 participates in the formation of the immunological synapse, which regulates T cell activation synergistically with TCR engagement. The immunological synapse is a specialized structure that forms between the T cell and the APC or target cell (1, 2, 4). The function of the immunological synapse is to facilitate T cell activation and signal transduction. Mice deficient in LFA-1 (CD11a KO) have defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection (5–7).Upon TCR stimulation, the nascent immunological synapse is initiated with surface receptor clustering and cytoskeleton rearrangement, then followed by mature synapse formation after prolonged stimulation (8, 9). In the mature immunological synapse, LFA-1 forms a ring-like pattern at the peripheral supramolecular activation cluster (pSMAC), which surrounds the central supramolecular activation cluster (cSMAC) containing TCR/CD3/lipid rafts (10, 11). The structure of the mature synapse is stable for hours and thought to be important for sustained TCR signaling (12–14). LFA-1 functions via pSMAC to stabilize the cSMAC and is associated with the induction of T cell proliferation, cytokine production, and lytic granule migration toward cSMAC (1, 15). Although LFA-1-containing pSMAC is self-evident in lipid bilayer systems and cell lines, whether it is required for T cell activation under physiological conditions remains controversial (15).TCR stimulation rapidly induces the functional activity of LFA-1, which then provides unique LFA-1-dependent signals to promote T cell activation (16). The process can be divided into two steps. First, the intracellular signaling from TCR regulating LFA-1 activation is known as “inside-out” signaling; second, activated LFA-1, as a signaling receptor, can feedback to transduce the intracellular signal, the “outside-in” signaling (1, 17). It is widely accepted that TCR stimulation activates LFA-1 through affinity and/or avidity regulation, as supported by increased adhesion to ICAM-1 and pSMAC formation (16, 17). The “inside-out” signal process has been investigated extensively (18–21). The TCR proximal signal molecules, Lck, ZAP-70, and PI3K, are known to be important for TCR signaling to LFA-1 activation (22–26). The molecular mechanisms of LFA-1 “outside-in” signaling have been explored only recently. Perez et al. (27) have demonstrated that LFA-1 and ICAM-1 ligation activates the downstream Erk1/2 MAPK signaling pathway upon TCR stimulation, which ultimately leads to the qualitative modulation of CD4⁺ T cell activation through distinct LFA-1-dependent signals. Another recent study provided compelling evidence that LFA-1 reshapes the Ras MAPK pathway downstream of TCR (28). However, the detailed molecular pathways that regulate these processes are poorly defined. Especially, the evidence in support of a distinctive role for LFA-1 in the T cell signaling pathway has lagged behind; whether the function of LFA-1 is to enhance TCR signaling through the immunological synapse and/or deliver distinct signal in T cell activation and whether LFA-1 is indispensable for or merely assists the existing TCR signal pathway. Furthermore, whether and how TCR proximal signal molecules regulate LFA-1 function remains unknown. Further studies are required to understand the LFA-1 and TCR signaling network.In this study, we found that LFA-1 directly participates in CD8⁺ T cell activation. Upon TCR stimulation, LFA-1 regulates both TCR-mediated and LFA-1-mediated Erk1/2 signal pathways. First, the presence of LFA-1, not ligand binding, is required for the sustained Erk1/2 signaling and TCR/CD3 clustering on the surface of CD8⁺ T cells, subsequently leading to MTOC reorientation, cell cycle progression, and mitosis. Second, LFA-1 ligation with ICAM-1 enhances Erk1/2 signaling, which promotes T cell activation with increased IL-2 production and cell proliferation. This LFA-1-mediated Erk1/2 signal pathway integrates with the existing TCR-mediated Erk1/2 signal pathway to enhance T cell activation.