Functional Characterization of the Dopaminergic Psychostimulant Sydnocarb as an Allosteric Modulator of the Human Dopamine Transporter.

The dopamine transporter (DAT) serves a critical role in controlling dopamine (DA)-mediated neurotransmission by regulating the clearance of DA from the synapse and extrasynaptic regions and thereby modulating DA action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DATs to alter their actions resulting in an enhancement in extracellular DA concentrations. We previously identified a novel allosteric site in the DAT and the related human serotonin transporter that lies outside the central orthosteric substrate- and cocaine-binding pocket. Here, we demonstrate that the dopaminergic psychostimulant sydnocarb is a ligand of this novel allosteric site. We identified the molecular determinants of the interaction between sydnocarb and DAT at the allosteric site using molecular dynamics simulations. Biochemical-substituted cysteine scanning accessibility experiments have supported the computational predictions by demonstrating the occurrence of specific interactions between sydnocarb and amino acids within the allosteric site. Functional dopamine uptake studies have further shown that sydnocarb is a noncompetitive inhibitor of DAT in accord with the involvement of a site different from the orthosteric site in binding this psychostimulant. Finally, DA uptake studies also demonstrate that sydnocarb affects the interaction of DAT with both cocaine and amphetamine. In summary, these studies further strengthen the prospect that allosteric modulation of DAT activity could have therapeutic potential.

Regulation of CFTR Bicarbonate Channel Activity by WNK1: Implications for Pancreatitis and CFTR-Related Disorders.

BACKGRAOUD & AIMS: Aberrant epithelial bicarbonate (HCO3-) secretion caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with several diseases including cystic fibrosis and pancreatitis. Dynamically regulated ion channel activity and anion selectivity of CFTR by kinases sensitive to intracellular chloride concentration ([Cl-]i) play an important role in epithelial HCO3- secretion. However, the molecular mechanisms of how [Cl-]i-dependent mechanisms regulate CFTR are unknown. METHODS: We examined the mechanisms of the CFTR HCO3- channel regulation by [Cl-]i-sensitive kinases using an integrated electrophysiological, molecular, and computational approach including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. In addition, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR. RESULTS: Among the WNK1, SPAK, and OSR1 kinases that constitute a [Cl-]i-sensitive kinase cascade, the expression of WNK1 alone was sufficient to increase the CFTR bicarbonate permeability (PHCO3/PCl) and conductance (GHCO3) in patch clamp recordings. Molecular dissection of the WNK1 domains revealed that the WNK1 kinase domain is responsible for CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions mediate the [Cl-]i-sensitivity of WNK1. Furthermore, the pancreatitis-causing R74Q and R75Q mutations in the elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation. CONCLUSION: The CFTR HCO3- channel activity is regulated by [Cl-]i and a WNK1-dependent mechanism. Our results provide new insights into the regulation of the ion selectivity of CFTR and the pathogenesis of CFTR-related disorders.

PINK1 Interacts with VCP/p97 and Activates PKA to Promote NSFL1C/p47 Phosphorylation and Dendritic Arborization in Neurons.

While PTEN-induced kinase 1 (PINK1) is well characterized for its role in mitochondrial homeostasis, much less is known concerning its ability to prevent synaptodendritic degeneration. Using unbiased proteomic methods, we identified valosin-containing protein (VCP) as a major PINK1-interacting protein. RNAi studies demonstrate that both VCP and its cofactor NSFL1C/p47 are necessary for the ability of PINK1 to increase dendritic complexity. Moreover, PINK1 regulates phosphorylation of p47, but not the VCP co-factor UFD1. Although neither VCP nor p47 interact directly with PKA, we found that PINK1 binds and phosphorylates the catalytic subunit of PKA at T197 [PKAcat(pT197)], a site known to activate the PKA holoenzyme. PKA in turn phosphorylates p47 at a novel site (S176) to regulate dendritic complexity. Given that PINK1 physically interacts with both the PKA holoenzyme and the VCP-p47 complex to promote dendritic arborization, we propose that PINK1 scaffolds a novel PINK1-VCP-PKA-p47 signaling pathway to orchestrate dendritogenesis in neurons. These findings highlight an important mechanism by which proteins genetically implicated in Parkinson's disease (PD; PINK1) and frontotemporal dementia (FTD; VCP) interact to support the health and maintenance of neuronal arbors.

Substrate transport and anion permeation proceed through distinct pathways in glutamate transporters.

Advances in structure-function analyses and computational biology have enabled a deeper understanding of how excitatory amino acid transporters (EAATs) mediate chloride permeation and substrate transport. However, the mechanism of structural coupling between these functions remains to be established. Using a combination of molecular modeling, substituted cysteine accessibility, electrophysiology and glutamate uptake assays, we identified a chloride-channeling conformer, iChS, transiently accessible as EAAT1 reconfigures from substrate/ion-loaded into a substrate-releasing conformer. Opening of the anion permeation path in this iChS is controlled by the elevator-like movement of the substrate-binding core, along with its wall that simultaneously lines the anion permeation path (global); and repacking of a cluster of hydrophobic residues near the extracellular vestibule (local). Moreover, our results demonstrate that stabilization of iChS by chemical modifications favors anion channeling at the expense of substrate transport, suggesting a mutually exclusive regulation mediated by the movement of the flexible wall lining the two regions.

Pore dilatation increases the bicarbonate permeability of CFTR, ANO1 and glycine receptor anion channels.

KEY POINTS: Cellular stimuli can modulate the ion selectivity of some anion channels, such as CFTR, ANO1 and the glycine receptor (GlyR), by changing pore size. Ion selectivity of CFTR, ANO1 and GlyR is critically affected by the electric permittivity and diameter of the channel pore. Pore size change affects the energy barriers of ion dehydration as well as that of size-exclusion of anion permeation. Pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of CFTR, ANO1 and GlyR. Dynamic change in P HC O3/ Cl may mediate many physiological and pathological processes. ABSTRACT: Chloride (Cl(-) ) and bicarbonate (HCO3 (-) ) are two major anions and their permeation through anion channels plays essential roles in our body. However, the mechanism of ion selection by the anion channels is largely unknown. Here, we provide evidence that pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of anion channels by reducing energy barriers of size-exclusion and ion dehydration of HCO3 (-) permeation. Molecular, physiological and computational analyses of major anion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR), anoctamin-1(ANO1/TMEM16A) and the glycine receptor (GlyR), revealed that the ion selectivity of anion channels is basically determined by the electric permittivity and diameter of the pore. Importantly, cellular stimuli dynamically modulate the anion selectivity of CFTR and ANO1 by changing the pore size. In addition, pore dilatation by a mutation in the pore-lining region alters the anion selectivity of GlyR. Changes in pore size affected not only the energy barriers of size exclusion but that of ion dehydration by altering the electric permittivity of water-filled cavity in the pore. The dynamic increase in P HC O3/ Cl by pore dilatation may have many physiological and pathophysiological implications ranging from epithelial HCO3 (-) secretion to neuronal excitation.

Energetics and ion permeation characteristics in a glutamate-gated chloride (GluCl) receptor channel.

An invertebrate glutamate-gated chloride channel (GluCl) has recently been crystallized in an open-pore state. This channel is homologous to the human Cys-loop receptor family of pentameric ligand-gated ion channels, including anion-selective GlyR and GABAR and cation-selective nAChR and 5HT(3). We implemented molecular dynamics (MD) in conjunction with an elastic network model to perturb the X-ray structure of GluCl and investigated the open channel stability and its ion permeation characteristics. Our study suggests that TM2 helical tilting may close GluCl near the hydrophobic constriction L254 (L9'), similar to its cation-selective homologues. Ion permeation characteristics were determined by Brownian dynamics simulations using a hybrid MD/continuum electrostatics approach to evaluate the free energy profiles for ion transport. Near the selectivity filter region (P243 or P-2'), the free energy barrier for Na(+) transport is over 4 k(B)T higher than that for Cl(-), indicating anion selectivity of the channel. Furthermore, three layers of positivity charged rings in the extracellular domain also contribute to charge selectivity and facilitate Cl(-) permeability over Na(+). Collectively, the charge selectivity of GluCl may be determined by overall electrostatic and ion dehydration effects, perhaps not deriving from a single region of the channel (the selectivity filter region near the intracellular entrance).

In silico models for the human alpha4beta2 nicotinic acetylcholine receptor.

The neuronal alpha4beta2 nicotinic acetylcholine receptor (nAChR) is one of the most widely expressed nAChR subtypes in the brain. Its subunits have high sequence identity (54 and 46% for alpha4 and beta2, respectively) with alpha and beta subunits in Torpedo nAChR. Using the known structure of the Torpedo nAChR as a template, the closed-channel structure of the alpha4beta2 nAChR was constructed through homology modeling. Normal-mode analysis was performed on this closed structure and the resulting lowest frequency mode was applied to it for a "twist-to-open" motion, which increased the minimum pore radius from 2.7 to 3.4 A and generated an open-channel model. Nicotine could bind to the predicted agonist binding sites in the open-channel model but not in the closed one. Both models were subsequently equilibrated in a ternary lipid mixture via extensive molecular dynamics (MD) simulations. Over the course of 11 ns MD simulations, the open channel remained open with filled water, but the closed channel showed a much lower water density at its hydrophobic gate comprised of residues alpha4-V259 and alpha4-L263 and their homologous residues in the beta2 subunits. Brownian dynamics simulations of Na+ permeation through the open channel demonstrated a current-voltage relationship that was consistent with experimental data on the conducting state of alpha4beta2 nAChR. Besides establishment of the well-equilibrated closed- and open-channel alpha4beta2 structural models, the MD simulations on these models provided valuable insights into critical factors that potentially modulate channel gating. Rotation and tilting of TM2 helices led to changes in orientations of pore-lining residue side chains. Without concerted movement, the reorientation of one or two hydrophobic side chains could be enough for channel opening. The closed- and open-channel structures exhibited distinct patterns of electrostatic interactions at the interface of extracellular and transmembrane domains that might regulate the signal propagation of agonist binding to channel opening. A potential prominent role of the beta2 subunit in channel gating was also elucidated in the study.