GLUT2 in pancreatic and extra-pancreatic gluco-detection

Detection of variations in blood glucose concentrations by pancreatic &#103 -cells and a subsequent appropriate secretion of insulin are key events in the control of glucose homeostasis. Because a decreased capability to sense glycemic changes is a hallmark of type 2 diabetes, the glucose signalling pathway leading to insulin secretion in pancreatic &#103 -cells has been extensively studied. This signalling mechanism depends on glucose metabolism and requires the presence of specific molecules such as GLUT2, glucokinase and the K ATP channel subunits Kir6.2 and SUR1. Other cells are also able to sense variations in glycemia or in local glucose concentrations and to modulate different physiological functions participating in the general control of glucose and energy homeostasis. These include cells forming the hepatoportal vein glucose sensor, which controls glucose storage in the liver, counterregulation, food intake and glucose utilization by peripheral tissues and neurons in the hypothalamus and brainstem whose firing rates are modulated by local variations in glucose concentrations or, when not protected by a blood-brain barrier, directly by changes in blood glucose levels. These glucose-sensing neurons are involved in the control of insulin and glucagon secretion, food intake and energy expenditure. Here, recent physiological studies performed with GLUT2 -/- mice will be described, which indicate that this transporter is ess ential for glucose sensing by pancreatic &#103 -cells, by the hepatoportal sensor and by sensors, probably located centrally, which control activity of the autonomic nervous system and stimulate glucagon secretion. These studies may pave the way to a fine dissection of the molecular and cellular components of extra-pancreatic glucose sensors involved in the control of glucose and energy homeostasis.     

Mechanosensitive channels of Corynebacterium glutamicum functioning as exporters of l-glutamate and other valuable metabolites

In the industrial l-glutamate production established on the use of Corynebacterium glutamicum, l-glutamate synthesized intracellularly is exported through mechanosensitive transmembrane channel proteins (MscCG and MscCG2) activated by the force-from-lipids. The involvement of MscCG2 in l-glutamate export by C. glutamicum was demonstrated in 2018; however, MscCG was previously found to be the major exporter of l-glutamate. Recent advances in research methods, such as development of the microbial patch clamp, revealed unique characteristics of MscCG, including its conductance, opening and closing thresholds, and gating hysteresis, as well as the significant effect of membrane lipids on the channel properties. In addition, the cryoelectron microscopic structure of Escherichia coli MscS, the canonical representative of the mechanosensitive channel family to which MscCG and MscCG2 belong, revealed its new membrane-interacting region, new position within the lipid bilayer, and hook lipids in a newly defined cavity between subunits. In this short review, the applications of bacterial mechanosensitive channels in the development of effective microbial cell factories, which will contribute to sustainable development, are discussed.     

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N₃-ATP) and 8-azidoadenosine 5′-monophosphate (8-N₃-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N₃-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N₃-ATP at ATP-binding site 2. The adenylate kinase active center probe P¹,P⁵-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.     

The Sulfonylurea Receptor 1 (Sur1)-Transient Receptor Potential Melastatin 4 (Trpm4) Channel

The sulfonylurea receptor 1 (Sur1)-NC_Ca-ATP channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NC_Ca-ATP channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and K_ATP channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.     

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains

The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.     

Extracellular Norepinephrine Clearance by the Norepinephrine Transporter Is Required for Skeletal Homeostasis

Changes in bone remodeling induced by pharmacological and genetic manipulation of β-adrenergic receptor (βAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.     

Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.     

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative,nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes

In the present study, one has determined the relative role of plasma membrane equilibrative (Na⁺-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na⁺-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2' 3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine)and ddI (2' 3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.     

Role of vacuolar transporter proteins in plant secondary metabolism: Catharanthus roseus cell culture

Here the current status of knowledge on some well-characterized transporters located in the vacuolar membrane is reviewed. As different cellular compartments and even different cells may be involved in certain steps of a biosynthetic pathway, the regulation of the flux is not only dependent on structural genes encoding enzymes catabolizing certain steps but also transport has a major regulatory function. The aim of the present review is to give an overview of the present knowledge of transport of secondary metabolites in plants, and to use this information in the context of our knowledge about Catharanthus roseus alkaloid biosynthesis. This should lead to further insight in the possible role of various transporters in the regulation of the biosynthesis of these alkaloids.