Thomas Stockner

Membrane-Mediated Effect on Ion Channels Induced by the Anesthetic Drug Ketamine

Anesthetic drugs have been in use for over 160 years in surgery, but their mode of action remains largely unresolved. We have studied the effect of (R)-(-)-ketamine on the biophysical properties of lipid model membranes composed of palmitoyloleoylphosphatidylcholine by a combination of X-ray diffraction and all-atom molecular dynamics simulations. In agreement with several previous studies, we do not find significant changes to the membrane thickness and lateral area per lipid up to 8 mol % ketamine content. However, we observed that the insertion of ketamine within the lipid/water interface caused significant changes of lateral pressure and a pressure shift toward the center of the bilayer. The changes are predicted to be large enough to affect the opening probability of ion channels as derived for two protein models. Depending on the protein model, we found inhibition values of IC(50) = 2 mol % and 18 mol % ketamine, corresponding to approximately 0.08 and 0.9 muM concentrations in the blood circulation, respectively. This compares remarkably well with clinical applied concentrations. We thus provide evidence for a lateral pressure mediated mode of anesthesia, first proposed more than 10 years ago.

Computational models for prediction of interactions with ABC-transporters

The polyspecific ligand recognition pattern of ATB-binding cassette (ABC)-transporters, combined with the limited knowledge on the molecular basis of their multispecificity, makes it difficult to apply traditional molecular modelling and quantitative structure-activity relationships (QSAR) methods for identification of new ligands. Recent advances relied mainly on pharmacophore modelling and machine learning methods. Structure-based design studies suffer from the lack of available protein structures at atomic resolution. The recently published protein homology models of P-glycoprotein structure, based on the high-resolution structure of the bacterial ABC-transporter of Sav1866, may open a new chapter for structure-based studies. Last, but not least, molecular dynamics simulations have already proved their high potential for structure-function modelling of ABC-transporter. Because of the recognition of several ABC-transporters as antitargets, algorithms for predicting substrate properties are of increasing interest.

The Mechanistic Basis for Noncompetitive Ibogaine Inhibition of Serotonin and Dopamine Transporters

Background: Ibogaine is a noncompetitive inhibitor of SERT that stabilizes the transporter in an inward-open conformation. Results: Ibogaine binds to a site accessible from the cell exterior that does not overlap with the substrate-binding site. Conclusion: Ibogaine binds to a novel binding site on SERT and DAT. Significance: This study provides a mechanistic understanding of an unique inhibitor of SERT and DAT. Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.

PKC-dependent coupling of calcium permeation through transient receptor potential canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes

Cardiac transient receptor potential canonical (TRPC) channels are crucial upstream components of Ca2+/calcineurin/nuclear factor of activated T cells (NFAT) signaling, thereby controlling cardiac transcriptional programs. The linkage between TRPC-mediated Ca2+ signals and NFAT activity is still incompletely understood. TRPC conductances may govern calcineurin activity and NFAT translocation by supplying Ca2+ either directly through the TRPC pore into a regulatory microdomain or indirectly via promotion of voltage-dependent Ca2+ entry. Here, we show that a point mutation in the TRPC3 selectivity filter (E630Q), which disrupts Ca2+ permeability but preserves monovalent permeation, abrogates agonist-induced NFAT signaling in HEK293 cells as well as in murine HL-1 atrial myocytes. The E630Q mutation fully retains the ability to convert phospholipase C-linked stimuli into L-type (CaV1.2) channel-mediated Ca2+ entry in HL-1 cells, thereby generating a dihydropyridine-sensitive Ca2+ signal that is isolated from the NFAT pathway. Prevention of PKC-dependent modulation of TRPC3 by either inhibition of cellular kinase activity or mutation of a critical phosphorylation site in TRPC3 (T573A), which disrupts targeting of calcineurin into the channel complex, converts cardiac TRPC3-mediated Ca2+ signaling into a transcriptionally silent mode. Thus, we demonstrate a dichotomy of TRPC-mediated Ca2+ signaling in the heart constituting two distinct pathways that are differentially linked to gene transcription. Coupling of TRPC3 activity to NFAT translocation requires microdomain Ca2+ signaling by PKC-modified TRPC3 complexes. Our results identify TRPC3 as a pivotal signaling gateway in Ca2+-dependent control of cardiac gene expression.

Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters

Highlights • We quantified adulterants in street drugs sold as cocaine.• We analyzed effects of the most common adulterant levamisole, on neurotransmitter transporters.• Differences in the selectivity of levamisole can be explained by homology modelling and docking.• Aminorex, a metabolite of levamisole, modulates neurotransmitter transporters directly.• Depending on the transporter, aminorex acts as a blocker or as a releaser.

Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

Unifying Concept of Serotonin Transporter-associated Currents

Background: hSERT is a neurotransmitter transporter driven by ion gradients with electroneutral stoichiometry but rheogenic properties. Results: hSERT displays coupled and uncoupled currents. The uncoupled current depends on internal K+. Conclusion: The conducting state of hSERT is in equilibrium with an inward facing K+-bound state. Significance: This study provides a framework for exploring transporter-associated currents. Serotonin (5-HT) uptake by the human serotonin transporter (hSERT) is driven by ion gradients. The stoichiometry of transported 5-HT and ions is predicted to result in electroneutral charge movement. However, hSERT mediates a current when challenged with 5-HT. This discrepancy can be accounted for by an uncoupled ion flux. Here, we investigated the mechanistic basis of the uncoupled currents and its relation to the conformational cycle of hSERT. Our observations support the conclusion that the conducting state underlying the uncoupled ion flux is in equilibrium with an inward facing state of the transporter with K+ bound. We identified conditions associated with accumulation of the transporter in inward facing conformations. Manipulations that increased the abundance of inward facing states resulted in enhanced steady-state currents. We present a comprehensive kinetic model of the transport cycle, which recapitulates salient features of the recorded currents. This study provides a framework for exploring transporter-associated currents.

‘Second-Generation’ Mephedrone Analogs, 4-MEC and 4-MePPP, Differentially Affect Monoamine Transporter Function

The nonmedical use of synthetic cathinones is increasing on a global scale. 4-Methyl-N-methylcathinone (mephedrone) is a popular synthetic cathinone that is now illegal in the United States and other countries. Since the legislative ban on mephedrone, a number of ‘second-generation’ analogs have appeared in the street drug marketplace, including 4-methyl-N-ethylcathinone (4-MEC) and 4′-methyl-α-pyrrolidinopropiophenone (4-MePPP). Here we characterized the interactions of 4-MEC and 4-MePPP with transporters for 5-HT (SERT) and dopamine (DAT) using molecular, cellular, and whole-animal methods. In vitro transporter assays revealed that 4-MEC displays unusual ‘hybrid’ activity as a SERT substrate (ie, 5-HT releaser) and DAT blocker, whereas 4-MePPP is a blocker at both transporters but more potent at DAT. In vivo microdialysis experiments in rat brain demonstrated that 4-MEC (1–3 mg/kg, i.v.) produced large increases in extracellular 5-HT, small increases in dopamine, and minimal motor stimulation. In contrast, 4-MePPP (1–3 mg/kg, i.v.) produced selective increases in dopamine and robust motor stimulation. Consistent with its activity as a SERT substrate, 4-MEC evoked inward current in SERT-expressing Xenopus oocytes, whereas 4-MePPP was inactive in this regard. To examine drug–transporter interactions at the molecular level, we modeled the fit of 4-MEC and 4-MePPP into the binding pockets for DAT and SERT. Subtle distinctions in ligand–transporter binding were found that account for the differential effects of 4-MEC and 4-MePPP at SERT. Collectively, our results provide key information about the pharmacology of newly emerging mephedrone analogs, and give clues to structural requirements that govern drug selectivity at DAT vs SERT.

Molecular Dissection of Dual Pseudosymmetric Solute Translocation Pathways in Human P-Glycoprotein

The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by high-resolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.

Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting.

Highlights ► Comparative oncological trials could speed up the development of novel therapies. ► Modeling revealed that human and canine ErbB-1 and ErbB-2 molecules are highly conserved. ► The canine ErbB molecules harbour epitopes for cetuximab and trastuzumab antibodies and indeed transmitted growth inhibitory signals. ► This knowledge opens up perspectives for comparative immunotherapy trials with canine cancer patients.