Inhibition of the norepinephrine transporter by the venom peptide c -MrIA: Site of action, Na+ dependence, and structure-activity relationship

c -MrIA and the NET. H a chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by replacement of Gly6. From this data we present a model of the structure of c -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.


Summary
χ-Conopeptide MrIA (χ-MrIA) is a thirteen 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 χ-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 µM) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action.

The binding of the NET inhibitors, [ 3 H]-nisoxetine and [ 3 H]-mazindol, to the expressed rat
and human NET was inhibited by χ-MrIA, with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by χ-MrIA was competitive in nature. It has previously been demonstrated that χ-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors, such as nisoxetine and mazindol, which do. This pattern of behaviour implies that the binding site for χ-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of χ-MrIA was found to be dependent on Na + , with the conopeptide becoming a less effective blocker of

Introduction
Because of its poor lipid solubility and degree of ionization at physiological pH, norepinephrine crosses cell membranes poorly by diffusion (1), and so relies on the operation of the norepinephrine transporter (NET 1 ) for uptake into cells. Clearance by this integral membrane protein constitutes the major mechanism for the termination of action of this neurotransmitter at noradrenergic synapses (2), and disturbances in the functioning of the NET are associated with pathological states including depression (3), congestive heart failure (4), and orthostatic intolerance and tachycardia (5). Known inhibitors of the NET include antidepressants (e.g., desipramine and nisoxetine), the appetite suppressant mazindol, and the abused drug cocaine (see review of 6). The NET, together with the dopamine transporter (DAT) and the serotonin transporter (SERT), form a family of Na + -and Cl-dependent monoamine transporters.  A novel peptidic NET inhibitor, χ-MrIA, has been identified in cone snail venom (7). Cone snails use a venom containing a cocktail of bioactive peptides (conopeptides) to capture their prey, and these are known to target an array of voltage-sensitive ion channels, ligand-gated ion-channels, and G protein-coupled receptors (for review, see 8). Intrathecal injection of χ-MrIA has been found to be analgesic in hot plate and neuropathic pain models (9,10). The inhibition of [ 3 H]-norepinephrine uptake by the NET caused by χ-MrIA was found to be non-competitive, reducing the maximum rate of transport and not affecting the transporters affinity for substrate (7). The non-competitive mode of action of χ-MrIA distinguishes it from the majority of the classically described inhibitors of the NET, which act in a competitive fashion. In this study, we explored the interaction of χ-MrIA with the monoamine transporters to gain an insight into the conopeptides selectivity, Na + dependence, site of action, and structure-activity relationship. by guest on July 9, 2020 http://www.jbc.org/ Downloaded from activation protocols (11) to couple the Fmoc-protected amino acid to the resin. The Fmoc protecting group was removed using 50% piperidine in dimethylformamide, and dimethylformamide was used as both the coupling solvent and for flow washes throughout the cycle. The progress of the assembly was monitored by quantitative ninhydrin monitoring (12).

Experimental Procedures
Peptide was deprotected and cleaved from the resin by stirring at room temperature in TFA:H 2 O:TIPS:EDT (90:5:2.5:2.5) for 2-3 hr. Cold diethyl ether was then added to the mixture and the peptide precipitated out. The precipitate was collected by centrifugation and subsequently washed with further cold diethyl ether to remove scavengers. The final product was dissolved in 50% aqueous acetonitrile and lyophilized to yield a fluffy white solid. The crude, reduced peptide was examined by reverse phase HPLC for purity, and the correct molecular weight confirmed by Electrospray mass spectrometry. Pure, reduced peptides were oxidized, and the major peak was purified to >95% purity, and characterized by HPLC prior to further use.  with TEM buffer and recentrifuged. The resulting pellet was resuspended in TEM buffer containing 10% glycerol. Rat brain homogenates were prepared as described previously (17).

Cellular uptake of [ 3 H]-monoamines-
Protein concentration was determined using the BCA protein assay kit (Pierce; Rockford, IL) . 9 by guest on July 9, 2020 http://www.jbc.org/ Downloaded from following the manufacturer's protocol. Aliquots of membrane were stored at -80°C until use.

Radioligand binding assays-
Binding reactions were set up in triplicate wells of 96-well plates. Membranes from COS-7 cells transfected with the rat or human NET (6 µg protein/well) were incubated with either 5 mM KCl, pH 7.4, for 1 hr at room temperature. The reactions were filtered, and the radioactivity counted, as described above.

H NMR spectroscopy-
All NMR spectra were recorded on a Bruker ARX 500 spectrometer equipped with a z-gradient unit. Peptide concentrations were ~2 mM. Each analog was examined in 95% H 2 O/5% The solvent was suppressed using the WATERGATE sequence (21). Spectra were processed using XWINMR. FIDs were multiplied by a polynomial function and apodised using a 90° shifted sine-bell function in both dimensions prior to Fourier transformation. Baseline correction using a 5 th order polynomial was applied. Chemical shift values were referenced internally to 2,2-dimethyl-2-silapetane-5-sulfonate (DSS) at 0.00 ppm. The peptides were assigned according to the method of Wüthrich (22). Secondary Hα shifts were compared to the random coil shift values of Wishart et al. (23).

Effect of χ-MrIA on the cellular uptake of [ 3 H]-monoamines
COS-1 cells transfected with either the rat or human NET readily accumulated Desipramine, nisoxetine and χ-MrIA reduced the binding of [ 3 H]-nisoxetine to rat brain homogenates in a concentration-dependent manner (Fig. 4). to the same extent (non-specific binding of ~43%), the estimated maximum extent of inhibition produced by χ-MrIA was significantly less (P < 0.001), with ~32% of the nisoxetine-and desipramine-sensitive binding found to be insensitive to χ-MrIA.

Sodium dependence of NET inhibition
The rate of uptake of [ 3 H]-norepinephrine by cells transfected with the human NET slowed substantially as the concentration of Na + in the transport buffer was reduced. At the lowest Na + concentration examined (25 mM) the rate of [ 3 H]-norepinephrine accumulation was approximately half of that observed at 125 mM Na + (data not shown). Concentrations of . 15 desipramine and χ-MrIA that inhibited transport by 50% in assays where the buffer contained 125 mM Na + (4.05 nM and 1.26 µM, respectively) were found to inhibit a progressively smaller proportion of the uptake in buffer containing less Na + (Fig. 5).

Structural effects of alanine substitutions
1D, TOCSY and NOESY 1 H NMR spectrum of χ-MrIA and analogs were recorded at 500 MHz and subsequently assigned using the sequential assignment protocol (22). Secondary chemical shifts, i.e. Hα chemical shifts compared to random coil values (25), are a sensitive measure of backbone conformation (26)(27)(28) and can provide an indication whether the overall global fold of a series of peptide is maintained (29). For a series of structurally related peptides, secondary Hα chemical shifts can be used to identify the location, but not the nature of local changes in conformation (29). Secondary Hα chemical shifts were used in the first instance to compare χ-MrIA with its alanine-substituted analogs (Fig. 7). The results indicate  7)) are shown in Figure 8.

Discussion
The aim of the present study was to investigate the influence transporter identity, the cosubstrate Na + , and individual residues of χ-MrIA have on the ability of the conopeptide to inhibit monoamine transporters. Whether χ-MrIA acted through a site on the NET that was distinct from classical inhibitors of the NET was also examined. χ-MrIA inhibited uptake by the NET of both species studied, and was found for expressed transporters to act with twice the potency at the rat over the human isoform. The amino acid sequence homology between the NETs of the two species is 93% (14). The NET is related to the transporters for the other monoamine neurotransmitters, dopamine and serotonin. The amino acid identity between the human NET and the human DAT is 66% (15), and between the human NET and human . 18 by guest on July 9, 2020 http://www.jbc.org/ Downloaded from SERT, the homology is 43% (16). Because a substantial number of inhibitors of the NET have relatively low specificity and also target the DAT, the SERT, or both (30) While the potency of χ-MrIA for inhibition of uptake and radioligand binding to the expressed rat NET observed here closely match its reported potency for potentiating noradrenergic contractions in the isolated rat vas deferens (430 nM; 7), we found that the potency of χ-MrIA for inhibition of the binding of [ 3 H]-nisoxetine to rat brain was an order of magnitude lower. Given its lower potency in the rat brain, its only partial inhibitory effect, and the modest degree of assumed specific (i.e., nisoxetine-or desipramine-sensitive) . 20 binding in the assay, it is perhaps not surprising that McIntosh et al. (9) did not detect any effect of χ-MrIA (10 µM) in their NET binding assay using conditions somewhat similar to those used here. A possible reason for the only partial inhibition of the specific [ 3 H]-nisoxetine binding by χ-MrIA is the additional binding of desipramine and nisoxetine to sites in the rat brain other than the NET, such as α 1 -adrenoceptors (44) or the SERT (30), which are not also targeted by χ-MrIA. Alternatively, the classical NET inhibitors may bind at multiple sites on the NET in a manner reminiscent of the interaction of the cocaine analog RTI-55 and the SERT (45), with χ-MrIA blocking only a subset of these. Our finding that χ-MrIA acts as a full inhibitor of the desipramine-sensitive [ 3 H]-nisoxetine binding to rat NETtransfected cell membranes ( Fig. 2A) discounts this hypothesis, or at least reflects a difference in the presentation of the NET in the membranes of native tissues and transfected cells, or even the existence of NET subtypes in the rat. The existence of such subtypes could explain the unexpected reduction in χ-MrIAs potency observed in the brain binding assay.
Norepinephrine transport by the NET has been shown to be dependent on Na + , reflecting the co-transport of Na + with the substrate (46). The reduced transport activity caused by lowering of the extracellular Na + concentration is mediated through an increase in the apparent K m for norepinephrine and a reduction in the V max . Extracellular Na + not only affects the transporter's affinity for substrate, but also its affinity for inhibitors. It has . 21 previously been shown that desipramine and other antidepressants become less effective inhibitors of uptake with reduced extracellular Na + (47), an observation confirmed in this study. χ-MrIA demonstrates the same pattern of Na + dependence. These findings may signify that desipramine and χ-MrIA target the outward-facing (substrate accessible) configuration of the transporter whose adoption is promoted by extracellular Na + (31). The Na + dependence of χ-MrIAs inhibitory action stands in contrast to that of cocaine, another natural product that inhibits the NET. Cocaine competes with Na + for binding to the NET, becoming a more potent inhibitor of transport as extracellular Na + decreases (47).
The three-dimensional structure of χ-MrIA has not been determined, but appears very similar to that of χ-MrIB (7), a conopeptide whose sequence differs by only a single residue at the N terminus and which displays very similar pharmacology to χ-MrIA. Like the majority of conopeptides, χ-MrIA contains multiple cysteine residues that are linked by intramolecular disulfide bonds. These bonds act to bring the cysteine pairs into close proximity in the core of the peptide, with residues in the intercysteine regions exposed as loops. Alanine scanning reveals a critical role for residues in the first and largest of χ-MrIAs two cysteine-bracketed loops in contributing to the peptides activity at the NET. Substitution of any of the residues in this region with alanine results in a loss of potency predicted to be in excess of ~600-fold. With the exception of the replacement of Gly6, the alanine substitutions did not affect the structure of the peptide backbone to any great degree. Tyr7, Lys8, Leu9, and Examination of the relative positions of the key binding determinants in χ-MrIA (Fig. 8) reveals a highly exposed Lys8 flanked by three hydrophobic residues to form the pharmacophore. Given its exposed position, it is possible that Lys8 could direct binding into a pore, perhaps reminiscent of how toxins such as charybdotoxin block the movement of K + ions through voltage-dependent channels (49). If this is indeed correct, as suggested by Bryan-Lluka et al. (43), χ-conopeptides might be useful molecular calipers to probe the size of the norepinephrine permeation pathway. The majority of transporter residues that have been found to influence tricyclic antidepressant binding lie in the predicted transmembrane domains of the NET (eg. 41,52,53). That the binding site of the antidepressants overlaps . with that of norepinephrine, yet χ-MrIAs binding site does not, is consistent with the χ-conopeptides acting to inhibit transport by blocking norepinephrines access to the substrate permeation pathway by binding less deeply in the transporters pore than do the classical small molecule inhibitors.
In summary, this study has shown that inhibition of monoamine transport by χ-MrIA is confined to that mediated by the NET. Accordingly, the χ-MrIA binding site on the NET seems likely to consist, at least partly, of residues that are not conserved between the NET and either the DAT or the SERT. Because χ-MrIA acts non-competitively with respect to norepinephrine, yet competitively with the classical NET inhibitors nisoxetine and mazindolwhich themselves are competitive inhibitors of norepinephrine uptakethe binding site of χ-MrIA is predicted to be distinct from the substrate binding site but to share some commonality with the antidepressant binding site. Furthermore, the Na + dependence exhibited by χ-MrIA indicates some similarity in its interaction with the NET to that of the antidepressants. Specific residues in loop one of χ-MrIA have been identified that directly interact with the NET or are important for the maintenance of a suitable peptide structure capable of recognizing the transporter. Further elucidation of how the χ-MrIA peptide binds to and inhibits the NET will reveal important structural and mechanistic information about the monoamine transporters which, at present, are poorly understood.