Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na ϩ -K ϩ -2Cl Ϫ cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (Ն100 M). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC 50 ϳ20 M, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC 50 ϳ8 M), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 M had no effect, and 20 M caused 20% block of human Na ϩ -K ϩ -2Cl Ϫ cotransporter activity, in contrast to Ͼ90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl Ϫ currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.
NKCC and KCC transporters mediate coupled transport of Na++K++Cl− and K++Cl− across the plasma membrane, thus regulating cell Cl− concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational analysis and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.
Background: Na-K-Cl cotransporters (NKCCs) are essential in chloride homeostasis and salt transport. Results: Mutations in NKCC1 transmembrane domain 3 (TM3) alter transport activity, ion binding, and inhibitor affinities. Conclusion: This demonstrates a role for TM3 in the NKCC1 transport pathway. Significance: This is the beginning of a systematic analysis of the Na-K-Cl cotransporter function in the context of structural models.
Background: Na-K-Cl cotransporters (NKCCs) are responsible for volume and chloride homeostasis and chloride transport and are targets of loop diuretic drugs. Results: Cross-links between transmembrane domains (TMs) 10 and 11/12 of NKCC1 are identified as inhibitory and stimulatory. Conclusion: Activation of NKCC1 involves movement of TM12 relative to TM10. Significance: This identifies movement of TM12 as a key step in the molecular mechanism of NKCC activation.
The Na-K-Cl cotransporter (NKCC) couples the movement of Na+, K+, and Cl− ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl− -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully “cys-less” NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.
NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces the barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells). Experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Epithelial cells derived from human intestine (T-84), airway (Calu-3), porcine intestine (IPEC-J2) and reproductive duct (PVD9902) were grown on permeable supports. Basal short circuit current (Isc) was <3 microA cm(-2) for T-84, IPEC-J2 and PVD9902 cell monolayers and<8 microA cm(-2) for Calu-3 cells. Apical NC-1059 exposure caused, in all cell types, an increase in Isc to >15 microA cm(-2), indicative of net anion secretion or cation absorption, which was followed by an increase in transepithelial conductance (in mS cm(-2): T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.3 to 13). These results are consistent with the peptide affecting transcellular ion movement, with a likely effect also on the paracellular route. NC-1059 exposure increased dextran permeation when compared to basal permeation, which documents an effect on the paracellular pathway. In order to evaluate membrane ion channels, experiments were conducted to study the dose dependence and stability of the NC-1059-induced membrane conductance in Xenopus laevis oocytes. NC-1059 induced a dose-dependent increase in oocyte membrane conductance that remained stable for greater than 2 h. The results demonstrate that NC-1059 increases transcellular conductance and paracellular permeation in a wide range of epithelia. These effects might be exploited to promote drug delivery across barrier epithelia.
(substrate and sodium ions in the core) were performed, in a fully atomistic, solvated bilayer environment. In the Apo_MD and the Na_MD simulations, the HP1 loop has a much higher mobility than the HP2 loop. However, the release of the substrate into the intracellular solvent in Asp_Na_MD, required the motions of both the HP1 and the HP2 loops. The opening up of HP2 loop facilitates solvation of the binding site resulting in the substrate being dislodged from its position. Prior to substrate release, the HP1 loop moves further down into the solvent, exposing the HP1-tip and the substrate to the solvent, followed by its subsequent release into the intracellular solvent. These results suggest that the intracellular gating involves sequential opening of both HP1 and HP2 loops. 1342-Pos Board B252Ligand Exit and Entry Pathways for Monoamine Transporters Bonnie A. Merchant, Jeffry D. Madura. The monoamine transporters are targets for various medicinal and illegal drugs that affect mood and behavior. Of particular interest are the dopamine (DAT) and serotonin (SERT) transporters of which the three-dimensional structures are unknown. A three-dimensional structure homologous to DAT and SERT, both in sequence and in function, is the leucine transporter (LeuT Aa ). While there is significant binding and uptake data, some structural information and homology models, there is no clear understanding of the transport pathways for ligands of LeuT Aa , DAT or SERT. The Random Acceleration Molecular Dynamics (RAMD) method as implemented in NAMD, was used to study the entry and exit pathways of various chemically relevant substrates in LeuT Aa and a homology model of DAT. Example pathways as illustrated in Figure 1. Free energy scores of the pathways have been characterized via the MultiConfiguration Thermodynamic Integration method. Several sites of low free energy score have been indentified, which correspond to primary and secondary substrate pockets of the transporters. Detailed free energy and structural results of the transport pathways will be presented. Figure 1. Representative transport pathways of leucine through LeuTAa using RAMD.
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