Patch-clamp and amperometric recordings from norepinephrine transporters: Channel activity and voltage-dependent uptake

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.

The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters

Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat49–57 (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat49–57 were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alanine-substituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat49–57 play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Tat49–57 in cellular uptake. In contrast, a 9-mer of l-arginine (R9) was 20-fold more efficient than Tat49–57 at cellular uptake as determined by Michaelis–Menton kinetic analysis. The d-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat49–57 play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this analysis, we designed and synthesized a class of polyguanidine peptoid derivatives. Remarkably, the subset of peptoid analogues containing a six-methylene spacer between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat49–57 and even to r9. Overall, a transporter has been developed that is superior to Tat49–57, protease resistent, and more readily and economically prepared.

Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. donovani (TUBA5) that is deficient in LdNT1 transport and consequently resistant to tubercidin. In TUBA5 cells, the LdNT1.2 genes had the same sequence as wild-type cells. However, because LdNT1.2 mRNA is not detectable in either wild-type or TUBA5 promastigotes, LdNT1.2 does not contribute to nucleoside transport in this stage of the life cycle. In contrast, the TUBA5 cells were compound heterozygotes at the LdNT1.1 locus containing two mutant alleles that encompassed distinct point mutations, each of which impaired transport function. One of the mutant LdNT1.1 alleles encoded a G183D substitution in predicted TM 5, and the other allele contained a C337Y change in predicted TM 7. Whereas G183D and C337Y mutants had only slightly elevated adenosine Km values, the severe impairment in transport resulted from drastically (≈20-fold) reduced Vmax values. Because these transporters were correctly targeted to the plasma membrane, the reduction in Vmax apparently resulted from a defect in translocation. Strikingly, G183 was essential for pyrimidine nucleoside but not adenosine transport. A mutant transporter with a G183A substitution had an altered substrate specificity, exhibiting robust adenosine transport but undetectable uridine uptake. These results suggest that TM 5 is likely to form part of the nucleoside translocation pathway in LdNT1.1

Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood–cerebrospinal-fluid drug-permeability barrier

The blood–brain barrier and a blood–cerebrospinal-fluid (CSF) barrier function together to isolate the brain from circulating drugs, toxins, and xenobiotics. The blood–CSF drug-permeability barrier is localized to the epithelium of the choroid plexus (CP). However, the molecular mechanisms regulating drug permeability across the CP epithelium are defined poorly. Herein, we describe a drug-permeability barrier in human and rodent CP mediated by epithelial-specific expression of the MDR1 (multidrug resistance) P glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP). Noninvasive single-photon-emission computed tomography with 99mTc-sestamibi, a membrane-permeant radiopharmaceutical whose transport is mediated by both Pgp and MRP, shows a large blood-to-CSF concentration gradient across intact CP epithelium in humans in vivo. In rats, pharmacokinetic analysis with 99mTc-sestamibi determined the concentration gradient to be greater than 100-fold. In membrane fractions of isolated native CP from rat, mouse, and human, the 170-kDa Pgp and 190-kDa MRP are identified readily. Furthermore, the murine proteins are absent in CP isolated from their respective mdr1a/1b(−/−) and mrp(−/−) gene knockout littermates. As determined by immunohistochemical and drug-transport analysis of native CP and polarized epithelial cell cultures derived from neonatal rat CP, Pgp localizes subapically, conferring an apical-to-basal transepithelial permeation barrier to radiolabeled drugs. Conversely, MRP localizes basolaterally, conferring an opposing basal-to-apical drug-permeation barrier. Together, these transporters may coordinate secretion and reabsorption of natural product substrates and therapeutic drugs, including chemotherapeutic agents, antipsychotics, and HIV protease inhibitors, into and out of the central nervous system.

Cocaine reward models: Conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.

Identification of an ATP-binding cassette transporter involved in bicarbonate uptake in the cyanobacterium Synechococcus sp. strain PCC 7942

Exposure of cells of cyanobacteria (blue–green algae) grown under high-CO2 conditions to inorganic C-limitation induces transcription of particular genes and expression of high-affinity CO2 and HCO3− transport systems. Among the low-CO2-inducible transcription units of Synechococcus sp. strain PCC 7942 is the cmpABCD operon, encoding an ATP-binding cassette transporter similar to the nitrate/nitrite transporter of the same cyanobacterium. A nitrogen-regulated promoter was used to selectively induce expression of the cmpABCD genes by growth of transgenic cells on nitrate under high CO2 conditions. Measurements of the initial rate of HCO3− uptake after onset of light, and of the steady-state rate of HCO3− uptake in the light, showed that the controlled induction of the cmp genes resulted in selective expression of high-affinity HCO3− transport activity. The forced expression of cmpABCD did not significantly increase the CO2 uptake capabilities of the cells. These findings demonstrated that the cmpABCD genes encode a high-affinity HCO3− transporter. A deletion mutant of cmpAB (M42) retained low CO2-inducible activity of HCO3− transport, indicating the occurrence of HCO3− transporter(s) distinct from the one encoded by cmpABCD. HCO3− uptake by low-CO2-induced M42 cells showed lower affinity for external HCO3− than for wild-type cells under the same conditions, showing that the HCO3− transporter encoded by cmpABCD has the highest affinity for HCO3− among the HCO3− transporters present in the cyanobacterium. This appears to be the first unambiguous identification and description of a primary active HCO3− transporter.

Genetic Separation of FK506 Susceptibility and Drug Transport in the Yeast Pdr5 ATP-binding Cassette Multidrug Resistance Transporter

Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.

Fluid-Phase Markers in the Basolateral Endocytic Pathway Accumulate in Response to the Actin Assembly-promoting Drug Jasplakinolide

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin–Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells.

Acute decrease in net glutamate uptake during energy deprivation

The extracellular glutamate concentration ([glu]o) rises during cerebral ischemia, reaching levels capable of inducing delayed neuronal death. The mechanisms underlying this glutamate accumulation remain controversial. We used N-methyl-d-aspartate receptors on CA3 pyramidal neurons as a real-time, on-site, glutamate sensor to identify the source of glutamate release in an in vitro model of ischemia. Using glutamate and l-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) as substrates and dl-threo-β-benzyloxyaspartate (TBOA) as an inhibitor of glutamate transporters, we demonstrate that energy deprivation decreases net glutamate uptake within 2–3 min and later promotes reverse glutamate transport. This process accounts for up to 50% of the glutamate accumulation during energy deprivation. Enhanced action potential-independent vesicular release also contributes to the increase in [glu]o, by ≈50%, but only once glutamate uptake is inhibited. These results indicate that a significant rise in [glu]o already occurs during the first minutes of energy deprivation and is the consequence of reduced uptake and increased vesicular and nonvesicular release of glutamate.

Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Recently, several SNARE proteins were found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homologue cellubrevin, and syntaxin-4. In this report we show that treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Furthermore, treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues.

Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes

The uptake and expression of extracellular DNA has been established as a mechanism for horizontal transfer of genes between bacterial species. Such transfer can support acquisition of advantageous elements, including determinants that affect the interactions between infectious organisms and their hosts. Here we show that erythrocyte-stage Plasmodium falciparum malaria parasites spontaneously take up DNA from the host cell cytoplasm into their nuclei. We have exploited this finding to produce levels of reporter expression in P.falciparum that are substantially improved over those obtained by electroporation protocols currently used to transfect malaria parasites. Parasites were transformed to a drug-resistant state when placed into cell culture with erythrocytes containing a plasmid encoding the human dihydrofolate reductase sequence. The findings reported here suggest that the malaria genome may be continually exposed to exogenous DNA from residual nuclear material in host erythrocytes.

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters

The Arabidopsis thaliana AtHKT1 protein, a Na+/K+ transporter, is capable of mediating inward Na+ currents in Xenopus laevis oocytes and K+ uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K+ transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K+ channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na+ currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli-alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135–142 and 377–384, face the cytosol, whereas the region of residues 55–62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.

Uptake of HCO3− and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta1

Mass-spectrometric disequilibrium analysis was applied to investigate CO2 uptake and HCO3− transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO2 (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO2 and HCO3−, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO3− was the dominant inorganic C species taken up, whereas HCO3− and CO2 were used at similar rates by C. reinhardtii. The apparent affinities of HCO3− transport and CO2 uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO2 and HCO3−. For chloroplasts from C. reinhardtii, the concentrations of HCO3− and CO2 required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO3− and CO2 transporters at the chloroplast envelope membrane.

Norepinephrine transporters have channel modes of conduction.

Neurotransmitter transporters couple to existing ion gradients to achieve reuptake of transmitter into presynaptic terminals. For coupled cotransport, substrates and ions cross the membrane in fixed stoichiometry. This is in contrast to ion channels, which carry an arbitrary number of ions depending on the channel open time. Members of the gamma-aminobutyric acid transporter gene family presumably function with fixed stoichiometry in which a set number of ions cotransport with one transmitter molecule. Here we report channel-like events from a presumably fixed stoichiometry [norepinephrine (NE)+, Na+, and Cl-], human NE (hNET) in the gamma-aminobutyric acid transporter gene family. These events are stimulated by NE and by guanethidine, an hNET substrate, and they are blocked by cocaine and the antidepressant desipramine. Voltage-clamp data combined with NE uptake data from these same cells indicate that hNETs have two functional modes of conduction: a classical transporter mode (T-mode) and a novel channel mode (C-mode). Both T-mode and C-mode are gated by the same substrates and antagonized by the same blockers. T-mode is putatively electrogenic because the transmitter and cotransported ions sum to one net charge. However, C-mode carries virtually all of the transmitter-induced current, even though it occurs with low probability. This is because each C-mode opening transports hundreds of charges per event. The existence of a channel mode of conduction in a previously established fixed-stoichiometry transporter suggests the appearance of an aqueous pore through the transporter protein during the transport cycle and may have significance for transporter regulation.

Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato.

Root hairs as specialized epidermal cells represent part of the outermost interface between a plant and its soil environment. They make up to 70% of the root surface and, therefore, are likely to contribute significantly to nutrient uptake. To study uptake systems for mineral nitrogen, three genes homologous to Arabidopsis nitrate and ammonium transporters (AtNrt1 and AtAmt1) were isolated from a root hair-specific tomato cDNA library. Accumulation of LeNrt1-1, LeNrt1-2, and LeAmt1 transcripts was root-specific, with no detectable transcripts in stems or leaves. Expression was root cell type-specific and regulated by nitrogen availability. LeNrt1-2 mRNA accumulation was restricted to root hairs that had been exposed to nitrate. In contrast, LeNrt1-1 transcripts were detected in root hairs as well as other root tissues under all nitrogen treatments applied. Analogous to LeNrt1-1, the gene LeAmt1 was expressed under all nitrogen conditions tested, and root hair-specific mRNA accumulation was highest following exposure to ammonium. Expression of LeAMT1 in an ammonium uptake-deficient yeast strain restored growth on low ammonium medium, confirming its involvement in ammonium transport. Root hair specificity and characteristics of substrate regulation suggest an important role of the three genes in uptake of mineral nitrogen.