A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft. The proteins belong to a large family of secondary transporters, which includes bacterial glutamate transporters. The C-terminal half of the glutamate transporters is well conserved and thought to contain the translocation path and the binding sites for substrate and coupling ions. A serine-rich sequence motif in this part of the proteins is located in a putative intracellular loop. Cysteine-scanning mutagenesis was applied to this loop in the glutamate transporter GltT of Bacillus stearothermophilus. The loop was found to be largely intracellular, but three consecutive positions in the conserved serine-rich motif (S269, S270, and E271) are accessible from both sides of the membrane. Single-cysteine mutants in the serine-rich motif were still capable of glutamate transport, but modification with N-ethylmaleimide blocked the transport activity in six mutants (T267C, A268C, S269C, S270C, E271C, and T272C). Two milimolars l-glutamate effectively protected against the modification of the cysteines at position 269–271 from the periplasmic side of the membrane but was unable to protect cysteine modification from the cytoplasmic side of the membrane. The results indicate that the conserved serine-rich motif in the glutamate transporter forms a reentrant loop, a structure that is found in several ion channels but is unusual for transporter proteins. The reentrant loop is of crucial importance for the function of the glutamate transporter.

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4–5 elementary charges.

Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins

The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest. We measured the immediate transcriptional response of yeast grown in rich media and treated with rapamycin to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways. The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle). Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways. Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p. Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p. The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule.

Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M1 muscarinic receptors, we activated the ATP-sensitive potassium (KATP) channel with a K+ channel opener and recorded the whole-cell KATP current. The KATP current was reversibly inhibited by the stimulation of the M1 receptor, which is linked to phospholipase C (PLC) by the Gq protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca2+ with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M1-receptor stimulation failed to inhibit the KATP current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP2 was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5′-[β,γ–imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the KATP channel was significantly higher when the M1 receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the KATP channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP2 levels.

Genetically determined chloride-sensitive hypertension and stroke

The stroke-prone spontaneously hypertensive rat (SHRSP) is a genetically determined model of “salt-sensitive” stroke and hypertension whose full phenotypic expression is said to require a diet high in Na+ and low in K+. We tested the hypothesis that dietary Cl− determines the phenotypic expression of the SHRSP. In the SHRSP fed a normal NaCl diet, supplementing dietary K+ with KCl exacerbated hypertension, whereas supplementing either KHCO3 or potassium citrate (KB/C) attenuated hypertension, when blood pressure (BP) was measured radiotelemetrically, directly and continually. Supplemental KCl, but not KB/C, induced strokes, which occurred in all and only those rats in the highest quartiles of both BP and plasma renin activity (PRA). PRA was higher with KCl than with KB/C. These observations demonstrate that with respect to both severity of hypertension and frequency of stroke the phenotypic expression of the SHRSP is (i) either increased or decreased, depending on whether the anionic component of the potassium salt supplemented is, or is not, Cl−; (ii) increased by supplementing Cl− without supplementing Na+, and despite supplementing K+; and hence (iii) both selectively Cl−-sensitive and Cl−-determined. The observations suggest that in the SHRSP selectively supplemented with Cl− the likelihood of stroke depends on the extent to which both BP and PRA increase.

Regulation of Ribosome Biogenesis by the Rapamycin-sensitive TOR-signaling Pathway in Saccharomyces cerevisiae

The TOR (target of rapamycin) signal transduction pathway is an important mechanism by which cell growth is controlled in all eucaryotic cells. Specifically, TOR signaling adjusts the protein biosynthetic capacity of cells according to nutrient availability. In mammalian cells, one branch of this pathway controls general translational initiation, whereas a separate branch specifically regulates the translation of ribosomal protein (r-protein) mRNAs. In Saccharomyces cerevisiae, the TOR pathway similarly regulates general translational initiation, but its specific role in the synthesis of ribosomal components is not well understood. Here we demonstrate that in yeast control of ribosome biosynthesis by the TOR pathway is surprisingly complex. In addition to general effects on translational initiation, TOR exerts drastic control over r-protein gene transcription as well as the synthesis and subsequent processing of 35S precursor rRNA. We also find that TOR signaling is a prerequisite for the induction of r-protein gene transcription that occurs in response to improved nutrient conditions. This induction has been shown previously to involve both the Ras-adenylate cyclase as well as the fermentable growth medium–induced pathways, and our results therefore suggest that these three pathways may be intimately linked.

Wortmannin-Sensitive Phosphorylation, Translocation, and Activation of PLCγ1, but Not PLCγ2, in Antigen-stimulated RBL-2H3 Mast Cells

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcεRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcεRI cross-linking activates both PLCγ1 and PLCγ2. Activation is accompanied by the increased phosphorylation of both PLCγ isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCγ isoforms have distinct subcellular localizations. PLCγ1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCγ1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCγ2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcεRI cross-linking. The activation of PLCγ1, but not of PLCγ2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCγ1 with little inhibition of PLCγ2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCγ1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCγ1. Although less abundant than PLCγ2, activated PLCγ1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.

Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2+ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2+; overexpression of atb2+ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

In Vitro Reconstitution of Microtubule Plus End-directed, GTPγS-sensitive Motility of Golgi MembranesV⃞

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5′-adenylylimidodiphosphate, and 100 μM but not 20 μM vanadate. Motility was also blocked by GTPγS or AlF4− but was insensitive to AlCl3, NaF, staurosporin, or okadaic acid. The targets for GTPγS and AlF4− were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.

Activation and Cellular Localization of the Cyclosporine A-sensitive Transcription Factor NF-AT in Skeletal Muscle Cells

The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT–mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT–responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.

The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel

Inwardly rectifying potassium (K+) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by Gβγ subunits released from G protein heterotrimers of the Gi/o family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein α subunits (Giα1–3 and GoαA) in mediating coupling between various receptor systems (A1, α2A, D2S, M4, GABAB1a+2, and GABAB1b+2) and the cloned counterpart of the neuronal channel (Kir3.1+3.2A). The expression of mutant PTx-resistant Gi/oα subunits in PTx-treated HEK293 cells stably expressing Kir3.1+3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A1, α2A) known to interact with all isoforms, Giα1–3 and GoαA can all support a significant degree of coupling to Kir3.1+3.2A. The M4 receptor appears to preferentially couple to Giα2 while another group of receptors (D2S, GABAB1a+2, GABAB1b+2) activates the channel predominantly through Gβγ liberated from GoA heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABAB1. Our data reveal selective pathways of receptor activation through different Gi/oα isoforms for stimulation of the G protein-gated inwardly rectifying K+ channel.

Determinant for β-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: An additional transmembrane region at the N terminus

The pore-forming α subunit of large conductance voltage- and Ca2+-sensitive K (MaxiK) channels is regulated by a β subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming α subunit necessary for β-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to β-subunit modulation, and analyzed the topology of the α subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel α subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1–S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers β-subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function.

Purification of the β-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells

The β cell-specific glucose-sensitive factor (GSF), which binds the A3 motif of the rat I and human insulin promoters, is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose only in normal islets, supporting the suggested physiological role of GSF in the glucose-regulated expression of the insulin gene. GSF binding activity was observed in all insulin-producing cells. We have therefore purified this activity from the rat insulinoma RIN and found that a single polypeptide of 45 kDa was responsible for DNA binding. Its amino acid sequence, determined by microsequencing, provided direct evidence that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as PDX-1) and that, in addition to its essential roles in development and differentiation of pancreatic islets and in β cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated β cells. The human cDNA coding for GSF/IPF-1 has been cloned, its cell and tissue distribution is described. Its expression in the glucagon-producing cell line αTC1 transactivates the wild-type human insulin promoter more efficiently than the mutated construct. It is demonstrated that high levels of ectopic GSF/IPF-1 inhibit the expression of the human insulin gene in normal islets, but not in transformed βTC1 cells. These results suggest the existence of a control mechanism, such as requirement for a coactivator of GSF/IPF-1, which may be present in limiting amounts in normal as opposed to transformed β cells.

Autoinhibition of serotonin cells: An intrinsic regulatory mechanism sensitive to the pattern of usage of the cells

After periods of high-frequency firing, the normal rhythmically active serotonin (5HT)-containing neurosecretory neurons of the lobster ventral nerve cord display a period of suppressed spike generation and reduced synaptic input that we refer to as “autoinhibition.” The duration of this autoinhibition is directly related to the magnitude and duration of the current injection triggering the high-frequency firing. More interesting, however, is that the autoinhibition is inversely related to the initial firing frequency of these cells within their normal range of firing (0.5–3 Hz). This allows more active 5HT neurons to resume firing after shorter durations of inhibition than cells that initially fired at slower rates. Although superfused 5HT inhibits the spontaneous firing of these cells, the persistence of autoinhibition in saline with no added calcium, in cadmium-containing saline, and in lobsters depleted of serotonin suggests that intrinsic membrane properties account for the autoinhibition. A similar autoinhibition is seen in spontaneously active octopamine neurons but is absent from spontaneously active γ-aminobutyric acid cells. Thus, this might be a characteristic feature of amine-containing neurosecretory neurons. The 5HT cells of vertebrate brain nuclei share similarities in firing frequencies, spike shapes, and inhibition by 5HT with the lobster cells that were the focus of this study. However, the mechanism suggested to underlie autoinhibition in vertebrate neurons is that 5HT released from activated or neighboring cells acts back on inhibitory autoreceptors that are found on the dendrites and cell bodies of these neurons.

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 107 reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.