Inhibition of the Norepinephrine Transporter by the Venom Peptide {chi}-MrIA: SITE OF ACTION, Na+ dependence, and structure-activity relationship

x-Conopeptide MrIA (-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether -MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 M) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [3H]nisoxetine and [3H]mazindol, to the expressed rat and human NET was inhibited by -MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by -MrIA was competitive in nature. It has previously been demonstrated that -MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for -MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of -MrIA was found to be dependent on Na with the conopeptide becoming a less effective blocker of [3H]norepinephrine by the NET under the conditions of reduced extracellular Na. In this respect, -MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of -MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of -MrIA and a His in loop 2 played a dominant role in the interaction between -MrIA and the NET. H chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of -MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET

The rat norepinephrine transporter: Molecular cloning from PC12 cells and functional expression

The rat norepinephrine transporter (rNET) cDNA from the PC12 pheochromocytoma cell line has been cloned by RT-PCR and characterized. The cDNA encodes an integral membrane protein consisting of 617 amino acids which contains twelve putative transmembrane domains, two potential N-glycosylation sites, two potential phosphorylation sites for protein kinase C and one phosphorylation site for casein kinase II. The nucleotide and deduced amino acid sequence shows a high level of homology to the human and the bovine norepinephrine transporter and less homology to the rat dopamine transporter (rDAT). Heterologous expression of rNET in HEK293 cells revealed that uptake of [H]norepinephrine is sodium- and chloride-dependent and highly sensitive to the selective norepinephrine transporter inhibitors desipramine and nisoxetine. The cloned rNET cDNA provides the opportunity to investigate this transporter in heterologous expression systems and adds a new member to the family of sodium- and chloride-dependent neurotransmitter transporters

Leveraging the n→π* Interaction in alkene isomerization by selective energy transfer catalysis

Examples of geometric alkene isomerization in nature are often limited to the net exergonic direction (ΔG°0) comparatively under-represented. Inspired by the expansiveness of the maleate to fumarate (Z→E) isomerization in biochemistry, we investigated the inverse E→Z variant to validate nO→πC=O* interactions as a driving force for contra-thermodynamic isomerization. A general protocol involving selective energy transfer catalysis with inexpensive thioxanthone as a sensitizer (λmax=402 nm) is disclosed. Whilst in the enzymatic process nO→πC=O* interactions commonly manifest themselves in the substrate, these same interactions are shown to underpin directionality in the antipodal reaction by shortening the product alkene chromophore. The process was validated with diverse fumarate derivatives (>30 examples, up to Z:E>99:1), including the first examples of tetrasubstituted alkenes, and the involvement of nO→πC=O* interactions was confirmed by X-ray crystallography