H⁺ Permeation and pH Regulation at a Mammalian Serotonin Transporter

The rat serotonin transporter expressed in Xenopusoocytes displays an inward current in the absence of 5-HT when external pH is lowered to 6.5 or below. The new current differs from the leakage current described previously in two ways. (1) It is ∼10-fold larger at pH 5 than the leakage current at pH 7.5 and reaches 1000 H⁺/sec per transporter at extremes of voltage and pH with no signs of saturation. (2) It is selective for H⁺ by reversal potential measurements. Similar H⁺-induced currents are also observed in several other ion-coupled transporters, including the GABA transporter, the dopamine transporter, and the Na⁺/glucose transporter. The high conductance and high selectivity of the H⁺-induced current suggest that protons may be conducted via a hydrogen-bonded chain (a “proton-wire mechanism”) formed at least partially by side chains within the transporter. In addition, pH affects other conducting states of rat serotonin transporter. Acidic pH potentiates the 5-HT-induced, transport-associated current and inhibits the hyperpolarization-activated transient current. The dose–response relationships for these two effects suggest that two H⁺ binding sites, with pK_a values close to 5.1 and close to 6.3, govern the potentiation of the 5-HT-induced current and the inhibition of the transient current, respectively. These results are important for developing structure-function models that explain permeation properties of neurotransmitter transporters

H⁺ Permeation and pH Regulation at a Mammalian Serotonin Transporter

The rat serotonin transporter expressed in Xenopusoocytes displays an inward current in the absence of 5-HT when external pH is lowered to 6.5 or below. The new current differs from the leakage current described previously in two ways. (1) It is ∼10-fold larger at pH 5 than the leakage current at pH 7.5 and reaches 1000 H⁺/sec per transporter at extremes of voltage and pH with no signs of saturation. (2) It is selective for H⁺ by reversal potential measurements. Similar H⁺-induced currents are also observed in several other ion-coupled transporters, including the GABA transporter, the dopamine transporter, and the Na⁺/glucose transporter. The high conductance and high selectivity of the H⁺-induced current suggest that protons may be conducted via a hydrogen-bonded chain (a “proton-wire mechanism”) formed at least partially by side chains within the transporter. In addition, pH affects other conducting states of rat serotonin transporter. Acidic pH potentiates the 5-HT-induced, transport-associated current and inhibits the hyperpolarization-activated transient current. The dose–response relationships for these two effects suggest that two H⁺ binding sites, with pK_a values close to 5.1 and close to 6.3, govern the potentiation of the 5-HT-induced current and the inhibition of the transient current, respectively. These results are important for developing structure-function models that explain permeation properties of neurotransmitter transporters

Cloning and Functional Expression of a Human Na + and Cl − -dependent Neutral and Cationic Amino Acid Transporter B 0+

A Na(+)-dependent neutral and cationic amino acid transport system (B(0+)) plays an important role in many cells and tissues; however, the molecular basis for this transport system is still unknown. To identify new transporters, the expressed sequence tag database was queried, and cDNA fragments with sequence similarity to the Na(+)/Cl(-)-dependent neurotransmitter transporter family were identified. Based on these sequences, rapid amplification of cDNA ends of human mammary gland cDNA was used to obtain a cDNA of 4.5 kilobases (kb). The open reading frame encodes a 642-amino acid protein named amino acid transporter B(0+). Human ATB(0+) (hATB(0+)) is a novel member of the Na(+)/Cl(-)-dependent neurotransmitter transporter family with the highest sequence similarity to the glycine and proline transporters. Northern blot analysis identified transcripts of approximately 4.5 kb and approximately 2 kb in the lung. Another tissue survey suggests expression in the trachea, salivary gland, mammary gland, stomach, and pituitary gland. Electrophysiology and radiolabeled amino acid uptake measurements were used to functionally characterize the transporter expressed in Xenopus oocytes. hATB(0+) was found to transport both neutral and cationic amino acids, with the highest affinity for hydrophobic amino acids and the lowest affinity for proline. Amino acid transport was Na(+) and Cl(-)-dependent and was attenuated in the presence of 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid, a system B(0+) inhibitor. These characteristics are consistent with system B(0+) amino acid transport. Thus, hATB(0+) is the first cloned B(0+) amino acid transporter

Membrane Transport Mechanisms Probed by Capacitance Measurements With Megahertz Voltage Clamp

We have used capacitance measurements with a 1-µs voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches, The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s^(-1) at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP-and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium, The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow 300- to 900-s^(-1), pump transition (the E_1-E_2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions, For the GAT1 (y-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which mag originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters

Leucine and arginine regulate trophoblast motility through mTOR-dependent and independent pathways in the preimplantation mouse embryo

AbstractUterine implantation is a critical element of mammalian reproduction and is a tightly and highly coordinated event. An intricate and reciprocal uterine-embryo dialog exists to synchronize uterine receptivity with the concomitant activation of the blastocyst, maximizing implantation success. While a number of pathways involved in regulating uterine receptivity have been identified in the mouse, less is understood about blastocyst activation, the process by which the trophectoderm (TE) receives extrinsic cues that initiate new characteristics essential for implantation. Amino acids (AA) have been found to regulate blastocyst activation and TE motility in vitro. In particular, we find that arginine and leucine alone are necessary and sufficient to induce TE motility. Both arginine and leucine act individually and additively to propagate signals that are dependent on the activity of the mammalian target of rapamycin complex 1 (mTORC1). The activities of the well-established downstream targets of mTORC1, p70S6K and 4EBP, do not correlate with trophoblast motility, suggesting that an independent-rapamycin-sensitive pathway operates to induce trophoblast motility, or that other, parallel amino acid-dependent pathways are also involved. We find that endogenous uterine factors act to induce mTORC1 activation and trophoblast motility at a specific time during pregnancy, and that this uterine signal is later than the previously defined signal that induces the attachment reaction. In vivo matured blastocysts exhibit competence to respond to an 8-hour AA stimulus by activating mTOR and subsequently undergoing trophoblast outgrowth by the morning of day 4.5 of pregnancy, but not on day 3.5. By the late afternoon of day 4.5, the embryos no longer require any exposure to AA to undergo trophoblast outgrowth in vitro, demonstrating the existence and timing of an equivalent in vivo signal. These results suggest that there are two separate uterine signals regulating implantation, one that primes the embryo for the attachment reaction and another that activates mTOR and initiates invasive behavior

Cell identity switching regulated by retinoic acid signaling maintains homogeneous segments in the hindbrain

© 2018 The Authors. Published by Elsevier. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1016/j.devcel.2018.04.003The patterning of tissues to form subdivisions with distinct and homogeneous regional identity is potentially disrupted by cell intermingling. Transplantation studies suggest that homogeneous segmental identity in the hindbrain is maintained by identity switching of cells that intermingle into another segment. We show that switching occurs during normal development and is mediated by feedback between segment identity and the retinoic acid degrading enzymes, cyp26b1 and cyp26c1. egr2, which specifies the segmental identity of rhombomeres r3 and r5, underlies the lower expression level of cyp26b1 and cyp26c1 in r3 and r5 compared with r2, r4, and r6. Consequently, r3 or r5 cells that intermingle into adjacent segments encounter cells with higher cyp26b1/c1 expression, which we find is required for downregulation of egr2b expression. Furthermore, egr2b expression is regulated in r2, r4, and r6 by non-autonomous mechanisms that depend upon the number of neighbors that express egr2b. These findings reveal that a community regulation of retinoid signaling maintains homogeneous segmental identity.This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK ( FC001217 ), the UK Medical Research Council ( FC001217 ), and the Wellcome Trust ( FC001217).Published versio

Amino Acid Residues that Control pH Modulation of TransportAssociated Current in Mammalian Serotonin Transporters

this paper (see Figs. 6, 7). TM, Transmembrane. B, Summary of the membrane topology shown in A. C, Alignments for 19 transporters, including #44 residues in the putative extracellular region studied in this paper, flanking the E493 region of rSERT. Initially, a total of 65 N

Permeation Properties of Neurotransmitter Transporters

The neurotransmitter transporters belong to three known families of intrinsic membrane proteins (Figure 1). The energy for transport, which is often against the neurotransmitter concentration gradient, is derived from the cotransport (and in some cases the counter transport) of inorganic ions. Ion-transporting ATPases establish the concentration gradients for these ions