Discovery of selective antagonists for the α9α10 nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChRs) are a class of ligand gated ion channels that are widely distributed in neuronal and non-neuronal cell types. One subtype of nAChRs, the α9α10 nAChR, is of particular interest as it has been implicated in pain signaling. Block of the α9α10 nAChR has demonstrated analgesia in several animal models. Because of the role of these receptors in pain, the discovery of antagonists of the α9α10 nAChR has important practical applications. Conus is a genus of venomous mollusks whose venom components have been widely utilized as pharmacological tools to discriminate between receptor and ion channel subtypes. Several species of Conus were selected and screened for activity against the α9α10 nAChR, with two of the venoms selected for further study. Subsequent purification led to the discovery of αS-GVIIIB, a novel σ-conotoxin that is potent for the α9α10 nAChR with an IC50 of 9.8 nM, and is over 100-fold more selective for the α9α10 nAChR compared to other nAChR subtypes. Furthermore, αS-GVIIIB gives increased insight into the σ-conotoxin family, a class of toxins that is not widely studied. Of particular interest is that the previously discovered σ-GVIIIA is selective for the 5-HT3 serotonin receptor and the newly discovered αS-GVIIIB, from the same Conus species, targets a different class of ligand-gated ion channels. The understanding of σ-conotoxins was also furthered with the demonstration that αS-GVIIIB competes with α-RgIA for the ACh binding domain, illustrating that αS-GVIIIB is a competitive antagonist. Also, toxins from the αD-conotoxin family are potent for the α9α10 nAChR, but selectivity is not a key feature as the αD-conotoxins are also potent for several other nAChRs.

DISCOVERY OF SELECTIVE ANTAGONISTS FOR THE α9α10 NICOTINIC ACETYLCHOLINE RECEPTOR by Sean Bradley Christensen A thesis submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Master of Science Department of Biology The University of Utah December 2016 Copyright © Sean Bradley Christensen 2016 All Rights Reserved The University of Utah Graduate School STATEMENT OF THESIS APPROVAL The thesis of Sean Bradley Christensen has been approved by the following supervisory committee members: J. Michael McIntosh , Chair 9/9/2014 Date Approved Grzegorz Bulaj , Member 9/9/2014 Date Approved Gary J. Rose , Member 9/9/2014 Date Approved and by , Chair/Dean of M. Denise Dearing the Department/College/School of and by David B. Kieda, Dean of The Graduate School. Biology ABSTRACT Nicotinic acetylcholine receptors (nAChRs) are a class of ligand gated ion channels that are widely distributed in neuronal and non-neuronal cell types. One subtype of nAChRs, the 910 nAChR, is of particular interest as it has been implicated in pain signaling. Block of the 910 nAChR has demonstrated analgesia in several animal models. Because of the role of these receptors in pain, the discovery of antagonists of the 910 nAChR has important practical applications. Conus is a genus of venomous mollusks whose venom components have been widely utilized as pharmacological tools to discriminate between receptor and ion channel subtypes. Several species of Conus were selected and screened for activity against the 910 nAChR, with two of the venoms selected for further study. Subsequent purification led to the discovery of S-GVIIIB, a novel -conotoxin that is potent for the 910 nAChR with an IC50 of 9.8 nM, and is over 100-fold more selective for the 910 nAChR compared to other nAChR subtypes. Furthermore, S-GVIIIB gives increased insight into the -conotoxin family, a class of toxins that is not widely studied. Of particular interest is that the previously discovered GVIIIA is selective for the 5-HT3 serotonin receptor and the newly discovered S-GVIIIB, from the same Conus species, targets a different class of ligand-gated ion channels. The understanding of -conotoxins was also furthered with the demonstration that S-GVIIIB competes with RgIA for the ACh binding domain, illustrating that S-GVIIIB is a competitive antagonist. Also, toxins from the D-conotoxin family are potent for the 910 nAChR, but selectivity is not a key feature as the D-conotoxins are also potent for several other nAChRs. TABLE OF CONTENTS ABSTRACT…………………………………………………….............…………………...……iii LIST OF TABLES………………………………………………………………………................v LIST OF FIGURES………………………………………………………..............……………...vi Chapters 1 INTRODUCTION……………………………............…………………………………...1 2 VENOM SCREENING…………………………............…………………………...……5 Introduction………………………………………............……………………….……….6 Methods………………………………………............……………………………………6 Results………………………………………............……..……....………...…………….7 Discussion……………………………………............………………………...………….7 3 αS-CONOTOXIN GVIIIB POTENTLY AND SELECTIVELY BLOCKS α9α10 NICOTINIC ACETYLCHOLINE RECEPTORS…………............…………..…10 Introduction…………………............……………………………………………............11 Materials and methods……………………............……………………………...............12 Results……………………………………............…………………………………........13 Discussion…………………………………............………………………………..........15 References………………………………............…………………………………….….17 4 CONUS CAPITANEUS…………………............……………………………............…19 Introduction……………………………………............………………............................20 Methods…………………………………………............……....................……………..20 Results……………………………………..............................…………………………..20 Discussion………………………………………............………………….................….21 5 CONCLUSION…………………………......................................................……………28 6 REFERENCES……………………………............…………….....................………….34 LIST OF TABLES 2.1 Venom screening on 910 nAChRs………………............……………………….…....9 3.1 Crude Conus venoms activity on α9α10 nAChRs…………........................................….13 3.2 αS-GVIIIB selectivity………………………………....................................................…16 3.3 α9α10 nAChR-targeting conotoxins…………………………..........................................16 3.4 Sequence alignment of venom purified σ-conotoxins………………...............................17 5.1 IC50 of rat versus human 910 nAChRs.........................................................................32 LIST OF FIGURES 3.1 HPLC purification of αS-GVIIIB……………............………………………….........….13 3.2 Sequence of αS-GVIIIB………………………………............………...............………..14 3.3 Concentration-response of native sigma-conotoxins…………............……….............…15 3.4 Activity of αS-GVIIIB on other neuronal nAChRs…………………...........................…15 3.5 Competition of α-RgIA and αS-GVIIIB for binding the α9α10 nAChR….......................16 3.6 Extracellular binding domain comparison of the 5-HT3 receptor and α9α10 nAChR………………………………………………………………............………...…17 4.1 Conus capitaneus fractions tested for block on 910 nAChRs......................................24 4.2 HPLC analysis of fraction 19.............................................................................................25 4.3 Activity of fraction 19 peak 1 following size-exclusion separation..................................26 4.4 HPLC hydrophobicity analysis of fraction 19 peak 1........................................................27 5.1 S-GVIIIB concentration-response curve for human 910 nAChRs.............................33 CHAPTER 1 INTRODUCTION 2 Introduction Background on Nicotinic Acetylcholine Receptors The nicotinic acetylcholine receptors (nAChRs) are a class of ligand gated ion channels that use acetylcholine (ACh) as their primary natural agonist. Each functional receptor is a pentamer containing five subunits; differing combinations of subunits determine the pharmacological role of the receptor [1]. There are seventeen known nAChR subunits; 2-10 and 2-4 are referred to as the neuronal subunits, and 1, 1, , ,  make up the muscle subtypes found at the neuromuscular junction. For the neuronal subtypes, typically a functional receptor is a heteromer comprised of both  and  subunits. There are also a few known functional homomers comprised of five subunits of 7 or 9 that form receptors, and 910 will form a receptor in the absence of a  subunit [2]. The 910 nAChR was originally identified in the cochlea as the receptor that mediates synaptic transmission between the olivocochlear efferents to auditory hair cells [3]. Subsequent studies have shown the presence of 910 nAChRs in non-neuronal tissues, including adrenal chromaffin cells, immune cells, and breast tumors [4] [5]. Several studies have demonstrated that block of 910 nAChRs is associated with analgesia [6] [7] [8]. Phylogenetically, the 9, 10, and 7 are considered the primordial receptor subunits, with the muscle subtypes coming next, and all the remaining neuronal subunits branching last [9]. Because of this relationship, the majority of antagonists that target 7, 910, or the muscle nAChR will have potency for all three subtypes. For example, a well-studied peptide -bungarotoxin from the venom of the banded krait snake Bungarus multicinctus potently blocks all three receptor subtypes. While the understanding of the role of the 910 nAChR is increasing, unfortunately, there are few available ligands available to characterize the function and pharmacology of 910 nAChRs. 3 Conotoxins as Neuropharmacological Tools The venom of predatory cone snails from the genus Conus have been extensively studied to yield new pharmacological tools that target a variety of ion channels with unique selectivity profiles [10]. There are approximately 500-700 species of Conus that use venom to prey upon worms, mollusks, and fish. Each individual species of venom is composed of roughly 100-200 peptide sequences that target different ion channels [11]. These toxin sequences have been grouped into families based upon peptide sequence similarities, cysteine framework, and the ion channel subtypes they target. Conotoxin Superfamilies that Target Nicotinic Acetylcholine Receptors Among the identified Conus toxin superfamilies, there are seven that have been shown to target nAChRs [12]. The most studied of these, the -conotoxins, are competitive blockers of the ACh binding site and have shown potency for several of the neuronal subtypes and the muscle subtype of nAChRs [13]. The A-conotoxins, also competitive for the ACh binding site, target mainly the muscle nAChR [14]. The C-conotoxins and the -conotoxins are two superfamilies that are noncompetitive blockers of the muscle nAChR [15] [16]. The S-conotoxins target nAChRs and have a preference for the muscle subtype, but are potent blockers of neuronal nAChRs [17]. It was previously unknown whether the S-conotoxins were competitive for the ACh binding site, but that was tested as part of this study. The D-conotoxins also target several neuronal nAChR subtypes; they are noncompetitive blockers that are not selective among subtypes as they will block several with high potency [18]. Finally, the recently discovered B3conotoxin VxXXIVA is a unique toxin that weakly, with about a ~1 M IC50, targets the 910 nAChR [19]. Of the seven superfamilies of conotoxins that target nAChRs, previous work has shown 4 only two of those to target the 910 nAChR. Several of the well-studied -conotoxins target the 910 nAChR as well as the aforementioned B3-VxXXIVA. Previously published conotoxins that target the 910 nAChR are -PeIA, -RgIA, and -Vc1.1 [20] [21] [7]. The first reported conotoxin antagonist of 910 nAChRs, -PeIA, was useful in that it discriminated between 910 and 7, but unfortunately, it was not selective for 910 as it was potent for several other neuronal subtypes. The next two characterized peptides, -RgIA and -Vc1.1, were both selective for the 910 nAChR. But a drawback for both peptides is that they have rapid off-rates for the 910 nAChR. Also, while both are selective and potent for the 910 nAChR, this testing was done for the rat 910 nAChR; when tested on the human 910 nAChR, the potency is significantly reduced [22] [23]. These two peptides have been instrumental in the study of the properties of the 910 nAChR, but most of these studies have been in rodents and an ideal toxin would be potent for both human and rat 910 nAChR. The scope of this project is to take a broad approach at discovering new antagonists of the 910 nAChR. The aforementioned previously identified antagonists were discovered by screening individual previously characterized compounds against the 910 nAChR. In an attempt to find additional antagonists using a broad approach, venoms from selected species of Conus were screened for activity against the 910 nAChR. With this method, roughly 100-200 compounds contained in each species venom can be screened together, and any venom that exhibits activity for the 910 nAChR can then be further studied to identify and characterize the active component(s) [24]. The advantage to this approach is that thousands of toxins can be screened in a short amount of time. The potential disadvantage is that the active component(s) could be hard to separate from the other venom components. Also, since the 910 nAChR is the target in this screening, there is also the potential that the active component(s) could have potent activity on other nAChR subtypes. CHAPTER 2 VENOM SCREENING 6 Introduction Seventeen venoms were initially selected for screening; these were selected from a variety of Conus clades representing all three major forms of predation (fish, mollusk, and worm) [25]. This would give a reasonable cross section of the genus Conus, and a wide variety of venom components should be present. The two desirable features are potency for the 910 nAChR, and a slow off-rate. Having a ligand with a slow off-rate would be a useful pharmacological tool that could be radiolabeled or tagged with a fluorescent dye to facilitate the visualization of the 910 nAChR in tissue preparations. Methods Preparation of Crude Venom Samples Small aliquot samples of whole venom of each species of Conus were prepared as follows. For each individual species, 40 mg of lyophilized venom ducts were weighed into a test tube and 800 L of B35 (65:35:0.1 H2O/acetonitrile/ trifluoroacetic acid (TFA)) was added. Next, the venom duct samples were homogenized by hand using a single-use disposable pestle, with the sample being ground a minimum of thirty rotations. The samples were then centrifuged and the supernatant was removed from the pellet. The supernatant of each species was then split into aliquots representing 2 mg of the original venom sample and lyophilized for future screening purposes. Oocyte Electrophysiology Oocytes were micro-injected with an equal ratio of cRNA of the 9 and 10 nAChR subtypes and allowed to incubate for 1-3 days. Oocytes expressing 910 nAChRs were voltage clamped at -70 mV using a two-electrode voltage clamp system (Warner Instruments) as previously described [26]. Briefly, the oocytes were placed in a 30 L bath and perfused with 7 ND96 (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, pH 7.5) containing 0.1 mg/mL bovine serum albumin (BSA). Venom samples were applied to the bath and allowed to incubate for 5 minutes. Following incubation, ACh was applied at a concentration of 10 M for 1 second followed by a 60 second wash, and the peak amplitude was recorded. For each species of Conus tested, initial screening started at 10 g of crude venom, and if over 80% block was observed, the sample was diluted ten-fold and tested again. Results Various Venoms Screened Whole venom preps were prepared as described and tested for activity against the 910 nAChR. Seventeen species were screened representing a broad array from the various Conus clades. Samples were initially screened at 10 g of venom extract per application (Table 2.1). Any sample that elicited greater than 80% block was diluted ten-fold to 1 g per application and tested again. For the 1 g per application, five species remained that blocked greater than 80% of the ACh-induced current. Of the five remaining species, four were from the same Conus clade, designated rhizoconus (C. vexillum, C. capitaneus, C. miles, and C. mustilinus), and the other species was C. geographus. When diluted ten-fold more to 100 ng of crude venom per application, only C. vexillum, C. capitaneus, and C. mustilinus blocked over 80% of the ACh response. When tested at 10 ng, C. capitaneus still blocked 78% of the ACh response. Discussion From the venom screening against the 910 nAChR, several candidates appeared promising for further study. For the five species that blocked greater than 80% per 1 g of venom application, all had a slow ACh response recovery after venom wash-out. Since four of the five species are closely related phylogenetically, they all belong to the rhizoconus clade, we 8 decided to select one species from this group for further investigation. Additionally, we decided to pursue the activity demonstrated by C. geographus, as this species is fairly diverse from the rhizoconus clade. The results from C. geographus are described in Chapter 3. From the rhizoconus clade, we selected C. capitaneus for further study because it was the most potent of the four species, and also because a peptide from C. vexillum, the aforementioned B3VxXXIVA, has a weak activity on the 910 nAChR [24]. The findings from C. capitaneus are presented in Chapter 4. 9 Table 2.1 Venom screening on 910 nAChRs % Block % Block % Block % Block Species SEM SEM 100 ng SEM 10 ng SEM 10 g 1 g C. vexillum 99.9 * 99.2 * 84.8 8.85 C. capitaneus 99.7 * 99.8 * 94.9 * 77.5 * C. mustelinus 99.8 * 99.8 * 99.4 * 56.9 * C. miles 99.2 * 88.8 * 16.2 7.00 C. rattus 92.4 * 13.4 4.58 C. imperialis 86.9 * C. caracteristicus 99.0 * 52.6 7.95 C. aulicus 46.7 1.44 C. quercinus 53.7 7.37 C. virgo 76.5 1.45 C. litteratus 15.7 4.58 C. geographus 98.8 0.38 84.7 3.42 5.2 2.75 C. brettinghami 91.0 1.21 13.8 2.41 C. radiatus 82.3 5.38 21.6 8.09 C. consors 96.1 0.79 38.1 6.58 C. ebraeus 50.2 * C. distans 29.7 * SEM, Standard Error of the Mean; *, less than 3 test applications. CHAPTER 3 αS-CONOTOXIN GVIIIB POTENTLY AND SELECTIVELY BLOCKS α9α10 NICOTINIC ACETYLCHOLINE RECEPTORS Sean B. Christensen, Pradip K. Bandyodpadhyay, Baldomero M. Olivera, and J. Michael McIntosh Reprinted with permission from Biochemical Pharmacology (2015) 96:349-356 Copyright © 2015 Elsevier Inc. 11 12 13 14 15 16 17 18 CHAPTER 4 CONUS CAPITANEUS 20 Introduction As mentioned in Chapter 2, the venoms screened from the rhizoconus clade all showed potent activity on the 910 nAChR. Conus capitaneus was selected for further workup, because its venom was the most potent of those in the clade, and showed long off-rate kinetics block. The start of the process was similar to that done for C. geographus, but sequence identification and selectivity testing was not performed after drawing conclusions from previously published work on the rhizoconus clade. Methods All the experimental procedures used for the Conus capitaneus venom are the same as those described for Conus geographus in Chapter 3. Oocyte testing and HPLC runs (both C18 and Size-exclusion) were performed in a similar manner as previously described. Results Purification of Conus capitaneus Venom A 200 mg venom extraction of Conus capitaneus was performed in a similar manner as described for Conus geographus in Chapter 3. The large-scale extraction was fractionated based on hydrophobicity using a preparative C18 column, and fractions were collected in 2 minute intervals. The fractions were then screened for activity using oocytes expressing the 910 nAChR. This screening revealed that fraction 19 contained the active component (Fig 4.1). Five percent of the active fraction was then analyzed by HPLC using a C18 column (Fig 4.2A). Inspection of the chromatogram indicated that there were multiple overlapping peaks, suggesting that the peptides may be difficult to efficiently resolve with reversed-phase chromatography. Ten percent of the fraction was therefore also analyzed by size-exclusion chromatography (Fig 4.2B), which produced three distinct peaks based on size. Using 910 nAChR expressing 21 oocytes, these three peaks were tested and peak one was the active component (Fig 4.3), while peaks two and three did not have any activity at the concentrations tested. Mass Spectrometry Analysis The three peaks separated by size in the active fraction were then submitted for MALDITOF mass spectrometry. The first peak (active fraction) had a mass of 10966.6 Da; peak two had a mass of 3928.9 Da; and the third peak had a mass of 2803.9 Da. Discussion D-conotoxins Are ~10 kDa Peptides that Non-Selectively Block Nicotinic Acetylcholine Receptors Previous studies have identified the D-conotoxin superfamily, a subset of conotoxins that target nAChRs. Loughnan et al. (2006) first identified the D-conotoxins from Conus vexillum, and described their activity against several nAChR subtypes [18]. Subsequently Loughnan et al. (2009) [27] identified several D-conotoxin sequences from similar Conus species through cDNA cloning techniques. This family of conotoxins consists of peptides that contain ten cysteine residues and are approximately 5000-5500 Da in size; the peptides form homodimers, doubling their mass to just over 10000 Da. To date, all the D-conotoxins identified are from the rhizoconus clade of Conus, specifically from the species C. vexillum, C. capitaneus, C. mustelinus, C. miles, and C. rattus [28]. The Active Component(s) of Conus capitaneus Venom Targeted to the a9a10 Nicotinic Acetylcholine Receptor May Be an aD-Conotoxin From this study, the venom of Conus capitaneus was fractionated and screened for activity on the 910 nAChR, resulting in an active component with a mass of 10966.6 Da; 22 although this is the largest intensity, there appears to be multiple other products. This mass is suggestive of the active component being an D-conotoxin. At this point, the decision was made to not pursue this active component for several reasons. First, the activity of D-conotoxins is potent, but fairly nonselective for nAChR subtypes. The D-conotoxins previously characterized have shown nanomolar potency for the 7 homomer and 2* containing subtypes (* - denotes any  subunit), and weak activity for 4* containing subtypes. It should be noted that the previous published studies of D-conotoxins were not screened for activity on 910 nAChRs. Although our screening of crude venom was done only on 910 nAChRs, Loughnan et al. reported the screening of C. capitaneus venom on several subtypes at 50 g per application. They reported complete block (100%) with a slow off-rate for 7, 32, and 42 nAChRs; and "the absence of significant inhibition" for 34 and 44. The sum of information suggests that the active component from C. capitaneus venom against the 910 nAChR is most likely not going to be subtype specific. It has been reported that the D-conotoxins are noncompetitive blockers of the ACh binding site, whereas in contrast, most -conotoxins and S-GVIIIB are competitive antagonists for the ACh binding site [27]. The other reason for not pursuing the active fraction is that separating the individual toxin components will be difficult due to multiple D-conotoxins present in the fraction. Loughnan et al. (2009) report five D-conotoxin sequences in the C. capitaneus venom from cDNA cloning [27]. They observed a mixture of peptides around 10,000 Da in mass present in their active fraction purified from venom [27]. This is consistent with our results from attempting to isolate the activity from the venom. As mentioned in the results, an attempt was made to separate the active fraction by size exclusion chromatography (Fig 4.2B). This was effective in creating a fraction that only contained peptides around 10000 Da in mass, but when analyzed using a C18 column (separation based on hydrophobicity), the results showed multiple peptides are still present (Fig 4.4). It is unknown how many D-conotoxins make up the active fraction, and if 23 one or more is responsible for the potent activity. The venom from Conus capitaneus elicited potent block of the 910 nAChR, and the recovery off-rate was slow. But upon investigation of the active component, the result was a large peptide, strongly assumed to be an D-conotoxin, that presents too many challenges in isolating and identifying the active component. Additionally, from published reports for screening on other nAChR subtypes, it is highly probable that the same active component(s) contributes to the block of multiple nAChR subtypes [18] [27]. 24 Figure 4.1 Conus capitaneus fractions tested for block on 910 nAChRs. From the 200 mg venom extraction, 10 ml fractions were collected based on hydrophobicity. For each application, a control response to 10 M ACh was established, labeled "C". After a baseline was established, ND96 flow was paused and one two-millionth of each fraction was allowed to incubate in the well containing an oocyte expressing 910 nAChR for 5 min. Subsequently, ND96 flow was resumed and the fraction was washed out. A. Fraction 19 was the most potent; fraction 18 and fraction 20 did not exhibit block greater than 10%, B. and C. 25 Figure 4.2 HPLC analysis of fraction 19. The active fraction was further analyzed using HPLC. A. Fraction 19 was first analyzed based on hydrophobicity using an analytical C18 column. One twentieth of the fraction was applied, and the gradient was 10% B60 (40:60:0.092 H2O/acetonitrile/trifluoroacetic acid) to 70% B60 for 60 min (1% per min) with a flow rate of 1 ml per min. B. Size-exclusion chromatography of fraction 19. One tenth of fraction 19 was applied, and peaks were separated based on mass. An isocratic gradient was used, with B30 (70:30:0.1 H2O/acetonitrile/trifluoroacetic acid). Peak 1 represents the active component(s). 26 Figure 4.3 Activity of fraction 19 peak 1 following size-exclusion separation. Xenopus laevis oocytes expressing 910 nAChRS were perfused with ND96. Once per min, control applications of 1 s ACh "C" were given, until a stable baseline was established. ND96 perfusion was stopped and one twenty-thousandth of fraction 19 peak 1 was applied. After 5 min of incubation, perfusion of the oocyte with ND96 was resumed and 1 s ACh pulses were applied every min. Responses were measured following sample washout. 27 Figure 4.4 HPLC hydrophobicity analysis of fraction 19 peak 1. Peak 1 collected from sizeexclusion separation exhibited potent activity when tested on 910 nAChRs. Peak 1 of fraction 19, with a mass of 10966.6 Da, was analyzed based on hydrophobicity a second time using a C18 column. One twentieth of the fraction was applied, and the gradient was 10% B60 (40:60:0.092 H2O/acetonitrile/trifluoroacetic acid) to 70% B60 for 60 min (1% per min) with a flow rate of 1 ml per min. Results indicate several components are still present. CHAPTER 5 CONCLUSION 29 Conclusion Broad Array of Conotoxin Superfamilies Active on 910 Nicotinic Acetylcholine Receptors Of the seven reported conotoxin superfamilies that have shown activity for nAChRs, only two superfamilies have previously been demonstrated to block the 910 nAChR. With this study, two more superfamilies that target nAChRs, the -conotoxins and the D-conotoxins, have been identified that potently block the 910 nAChR. S-GVIIIB Is Potent for Human 910 Nicotinic Acetylcholine Receptors As discussed above, the 910 nAChR is one of the primordial nAChRs along with the 7 and muscle subtypes. There has been some genetic drift between 910 nAChRs contained in the human versus the rat receptor [29]. Of the previously identified conotoxins that target 910 nAChRs, -RgIA and -Vc1.1 have both shown a significant loss in potency for the human receptor versus the rat receptor (Table 5.1) [22] [23]. As part of the extensive research with these peptides, key residues involved in the ACh binding site have been identified as critical for the loss in activity across species. As was demonstrated in Chapter 3, S-GVIIIB is competitive with -RgIA for the ACh binding site for the 910 nAChR. The activity of SGVIIIB was assessed for the human 910 nAChR, with an IC50 of 33.6 (26.6-42.3) nM (Fig 5.1). Thus, S-GVIIIB is potent on both the human and rat RgIA 910 nAChR. In contrast, RgIA has a much lower relative potency for the human vs. rat 910 nAChR (Table 5.1). Since RgIA is a competitive antagonist and prevents the binding of S-GVIIIB, the two toxins most likely bind to overlapping residues near the ACh binding domain of the rat 910 nAChR [30]. This suggests that one potential avenue for further study of S-GVIIIB is to identify the key residues near the ACh binding domain that are critical, and determine if these residues are conserved across species. Having another antagonist that binds to a site that overlaps with RgIA 30 but with slow off-rate kinetics has the potential to further the understanding of the 910 nAChR. conotoxins Have Diverse Receptor Targets With this report of the characterization and activity profile of S-GVIIIB, there are now three known targets of -conotoxins. Previous work identified -GVIIIA, specific for the serotonin 5-HT3 receptor and S-RVIIIA, a peptide that preferentially blocks the muscle nAChR along with potent activity on several other subtypes [31] [17]. With S-GVIIIB being selective for the 910 nAChR, the known targets of the -conotoxins are widening. Although the activity of these three peptides have been described, there is still much that is not known about the -conotoxins. All three of these peptides have been isolated from venom, with the oxidation of the disulfide bridges occurring in vivo. One advantage and the reason the -conotoxins are well characterized is because they contain 4 Cys with a known disulfide pattern that can be routinely synthesized using solid phase synthesis resulting in peptides that are similar to conotoxins purified from the venom. The -conotoxin superfamily is defined by the sequence spacing of 10 Cys residues, but the disulfide pattern is not known. Identifying the disulfide framework is a logical next step in advancing the understanding of the -conotoxins. This can facilitate the synthesis of -conotoxins in vitro, utilizing solid phase peptide synthesis. Successfully making a synthetic peptide that is similar in activity to one of the venom isolated compounds would be instrumental in establishing protocols that could be used for the several conotoxin sequences that have been identified from genetic data. New Activity Described for D-conotoxins The D-conotoxins have previously been reported as noncompetitive blockers of several nAChR subtypes [18]. This study suggests that the D-conotoxins are potent for910 nAChRs 31 in addition to the many other nAChRs they target. Although not definitively shown, there is strong suggestive evidence, based on mass and hydrophobicity profile, that the active component(s) of the venom is an D-conotoxin. The rhizoconus clade is unique in that there have not been any reported -conotoxins from the six species that are part of this group. With the absence of -conotoxins, the major class of conotoxins that target nAChRs in some species, the rhizoconus clade has utilized the D-conotoxins to target the nAChRs in their prey [28]. This illustrates the importance that Conus has placed on having components in the venom that potently block nAChRs. 32 Table 5.1 IC50 of rat versus human 910 nAChRs -RgIA -Vc1.1 S-GVIIIB Rat 910 1.49 nM 70.0 nM 9.79 nM Human 910 494 nM 975.4 nM 33.6 nM Ratio 331.5 13.9 3.4 Reference [22] [32] This Study 33 Figure 5.1 S-GVIIIB concentration-response curve for human 910 nAChRs. S-GVIIIB was tested on Xenopus laevis oocytes microinjected with an equal ratio of human 9 and human 10 nAChR subunits. S-GVIIIB blocked the human 910 nAChR with an IC50 of 33.6 (26.642.3) nM and a Hill slope of 1.5 (0.9-2.0). For the 3 nM, 10 nM, and 30 nM data points, a solution containing the toxin was perfused with 1 s ACh applications occurring once per min, until an equilibrium of block was achieved. 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