Society for Neuroscience Abstracts available at http://www.sfn.org/am2007/index.cfm?pagename=call_for_abstracts

*M. ZHANG1, M. HOLFORD1, P. CATLIN1, L. AZAM1, B. FIEDLER1, B. R. GREEN2, M. WATKINS1, B. M. OLIVERA1, G. BULAJ2, D. YOSHIKAMI1;
Novel conopeptides from Conus bullatus block mammalian voltage-gated sodium channels
1Dept Biol, 2Dept Medicinal Chem., Univ. Utah, Salt Lake City, UT

Abstract: We have discovered three new m-conopeptides, m-BuIIIA, m-BuIIIB, and m-BuIIIC, from the fish-hunting cone snail Conus bullatus by an exogenomics strategy (Olivera, J. Biol. Chem. 281, 31173) involving a combination of phylogenetics, molecular biology and chemical syntheses (Holford et al., Am. Pep. Soc. Symp. Abstract #292). Here, we report on the functional activities of these peptides. Preliminary experiments indicated that these peptides could block TTX-sensitive sodium currents in mouse DRG neurons as well as inhibit A-fiber compound action potentials in mouse sciatic nerve. We therefore examined the specificities of these peptides against seven cloned mammalian Na channels, NaV1.1 through 1.7, expressed in Xenopus oocytes and assessed by two-electrode voltage clamp experiments. At a concentration of 1 mM, BuIIIB and BuIIIC each blocked all seven channel isoforms to varying extent, with NaV1.1, 1.2, 1.3, and 1.4 being the most susceptible (³ 80% block). The block of NaV1.1, 1.2, and 1.3 by BuIIIB was irreversible, whereas BuIIIC irreversibly blocked only NaV1.2. BuIIIA was the least active of the three peptides: at 1 mM, it reversibly blocked the currents of NaV1.2, 1.3 and 1.4 and was inactive against NaV1.1, 1.6, and 1.7. All three bullatus peptides blocked NaV1.5 (the cardiac subtype) poorly (< 50% block at 1 mM). Of the three conopeptides, BuIIIC was the most effective in blocking NaV1.7 (~ 80% inhibition at 1µM), which is found in sensory neurons involved in communicating acute and inflammatory pain. In summary, C. bullatus m-conopeptides have unique functional activities when compared to each other and to other m-conopeptides. Thus, these newly identified conopeptides are valuable additions to the toolkit of probes for voltage-gated sodium channels.

*P. CHEN1, A. DENDORFER2, H. TERLAU2, B. OLIVERA1;
Conus peptides targeted to K channels: kM-conotoxins and cardioprotection
1Biol., Univ. Utah, Salt Lake City, UT; 2Inst. of exp. and clin. Pharmacol. and Toxicology, Luebeck, Germany

Abstract: Cone snail (Conus) venoms contain a large number of small, conformationally constrained peptides (conopeptides) that display highly potent and specific biological activity. Some conotoxins are useful ligands to identify distinct subtypes of ion channels and investigate different states and transitions between these states of the targeted ion channels. A set of structurally and genetically divergent Conus peptide families have been shown to target K channels in different groups of Conus species (1-3), including an O-conotoxin, k-PVIIA from Conus purpurascens, an M-conotoxin, kM-RIIIK from Conus radiatus, an A-conotoxin, kA-SVIA from Conus striatus, and conkunitzin-S1 from Conus striatus, a member of a Kunitz-type conotoxin family. These peptides were purified from fish-hunting snails and blocked the Kv1 (or Shaker) channel subfamily. Recently, we purified another kM-conotoxin, kM-RIIIJ, from a fish-hunting snail Conus radiatus that has high sequence similarity to kM-RIIIK, which also blocks human Kv1.2 subtype. Electrophysiological experiments show that kM-RIIIJ exhibits higher affinity on the human Kv1.2 channel expressed in the Xenopus oocytes than kM-RIIIK (IC50 of kM-RIIIK: 352 nM; IC50 of kM-RIIIJ: 33 nM). However, in an animal cardioprotective model, kM-RIIIK was cardioprotective and reduced the ishemia/repurfusion-induced infarction (4), whereas kM-RIIIJ did not exert any apparent cardioprotection. (Supported by NIH grant GM 48677.) 1. Terlau & Olivera. (2003) Physiol. Rev. 84:41. 2. Ferber et al. (2003) J. Biol. Chem. 278:2177. 3. Bayrhuber et al. (2005) J. Biol. Chem. 280:23766. 4. Terlau H., Olivera B. M., Dendorfer A. Joint Meeting of The German Society of Physiology and The Federation of European Physiologicall Societies. Oral session OM1-5.

*T. BORDIA1, S. R. GRADY2, J. M. MCINTOSH3, M. QUIK1;*T. BORDIA1, S. R. GRADY2, J. M. MCINTOSH3, M. QUIK1;
Nigrostriatal damage preferentially decreases a subpopulation of alpha6beta2* nAChRs in mouse, monkey and Parkinson's disease striatum
1Basic Res., The Parkinson's Inst., Sunnyvale, CA; 2Inst. Behav. Genet., Univ. Colorado, Boulder, CO; 3Dept. Biol. Psych., Univ. Utah, Salt Lake City, UT

Abstract: Parkinson’s disease is a neurodegenerative movement disorder characterized by a loss of substantia nigra dopamine neurons, and corresponding declines in molecular components present on striatal dopaminergic nerve terminals. These include the α6β2* nicotinic receptors (nAChRs), which are localized exclusively on dopamine terminals in striatum (*denotes the presence of possible additional subunits in the receptor complex). Here, we used a novel α-conotoxin MII analog E11A to further investigate α6β2* nAChR subtypes in mouse, monkey and human striatum. Receptor competition studies with 125I-α-conotoxin MII showed that E11A inhibition curves were biphasic, suggesting the presence of two distinct α6β2* nAChR subtypes. These include a very high (fM) and a high (pM) affinity site, with ~40% of the sites in the very high affinity form. Interestingly, only the high affinity form was detected in α4 nAChR null mutant mice. Since 125I-α-conotoxin MII binds primarily to α6α4β2β3 and α6β2β3 nAChR subtypes in mouse striatum, these data suggest that the population lost in the α4 knockout mice was the α6α4β2β3 subtype. We next investigated the effect of nigrostriatal lesioning on these two striatal α6β2* populations in two animal models and in Parkinson’s disease. There was a preferential loss of the very high affinity subtype in striatum of MPTP-treated mice, MPTP-treated monkeys and Parkinson’s disease cases. These data suggest that dopaminergic terminals expressing the α6α4β2β3 population are selectively vulnerable to nigrostriatal damage. This latter nAChR subtype, identified with α-conotoxin MII E11A, may therefore provide a unique marker for dopaminergic terminals particularly sensitive to nigrostriatal degeneration in Parkinson’s disease.

*P. WHITEAKER1, M. J. MARKS1, A. C. COLLINS1, J. M. MCINTOSH2;
[125I]α-conotoxin ArIB[V11L;V16A], a highly selective and reversible α7 subtype nicotinic acetylcholine receptor (nAChR) ligand
1Inst. for Behav Genentics, Boulder, CO; 2Biol. and Psychiatry, Univ. of Utah, Salt Lake City, UT

Abstract: α7 nAChRs are widely expressed in vertebrates, both in the central nervous system (CNS) and periphery. In the mammalian CNS, [125I]α-bungarotoxin (α-Bgt) is commonly used to identify α7 nAChRs specifically. However, α-Bgt also interacts potently with a8* nAChRs found in avian CNS, and with a1* and α9α10 nAChRs. [3H]Methyllycaconitine (MLA) is also frequently used as an α7-selective antagonist, but has significant affinity for α6* and α9α10 nAChRs subtypes. Previously, we described the cloning of a novel conotoxin (ArIB; from Conus arenatus) with relatively high selectivity for α7 nAChRs, and the generation, by directed substitution, of a series of α-CtxArIB derivatives with even greater α7 specificity. Here, we have developed a highly α7 selective radioligand by iodination of a naturally-occurring histidine residue within the previously-characterized α-conotoxin ArIB[V11L;V16A] analog. Both mono- and di-iodo derivatives were generated (specific activities were 2200 and 4400 Ci mmol-1, respectively). Iodinated derivatives were separated from each other and from unreacted precursors (free 125I and unlabled peptide) by reverse-phase HPLC. The properties of the mono- and di-iodo derivatives were extremely similar to each other. Saturation binding to mouse hippocampal membranes demonstrated a Kd of 1.15 ± 0.13 nM, similar to that of [125I]α-Bgt in the same preparations (0.52 ± 0.16 nM). Association and dissociation kinetics were relatively rapid (kobs for association at 1 nM was 0.024 ± 0.002 min-1; koff = 0.0195 ± 0.001 min-1). The selectivity of [125I]α-conotoxin ArIB[V11L;V16A] was confirmed in autoradiographic experiments using α7-null mutant tissue: specific binding was abolished in all regions of α7-/- brains, while wild-type mice expressed high levels of labeling and low non-specific binding. [125I]α-conotoxin ArIB[V11L;V16A] should prove useful where α7 nAChRs are co-expressed with other subtypes which are also labeled by the existing ligands [125I]α-Bgt and / or [3H]MLA. Further, true equilibrium binding experiments could be performed on α7 nAChRs, something that is impossible with [125I]α-Bgt.

*P. WHITEAKER1, M. J. MARKS1, A. C. COLLINS1, J. M. MCINTOSH2;
[125I]α-conotoxin ArIB[V11L;V16A], a highly selective and reversible α7 subtype nicotinic acetylcholine receptor (nAChR) ligand
1Inst. for Behav Genentics, Boulder, CO; 2Biol. and Psychiatry, Univ. of Utah, Salt Lake City, UT

Abstract: α7 nAChRs are widely expressed in vertebrates, both in the central nervous system (CNS) and periphery. In the mammalian CNS, [125I]α-bungarotoxin (α-Bgt) is commonly used to identify α7 nAChRs specifically. However, α-Bgt also interacts potently with a8* nAChRs found in avian CNS, and with a1* and α9α10 nAChRs. [3H]Methyllycaconitine (MLA) is also frequently used as an α7-selective antagonist, but has significant affinity for α6* and α9α10 nAChRs subtypes. Previously, we described the cloning of a novel conotoxin (ArIB; from Conus arenatus) with relatively high selectivity for α7 nAChRs, and the generation, by directed substitution, of a series of α-CtxArIB derivatives with even greater α7 specificity. Here, we have developed a highly α7 selective radioligand by iodination of a naturally-occurring histidine residue within the previously-characterized α-conotoxin ArIB[V11L;V16A] analog. Both mono- and di-iodo derivatives were generated (specific activities were 2200 and 4400 Ci mmol-1, respectively). Iodinated derivatives were separated from each other and from unreacted precursors (free 125I and unlabled peptide) by reverse-phase HPLC. The properties of the mono- and di-iodo derivatives were extremely similar to each other. Saturation binding to mouse hippocampal membranes demonstrated a Kd of 1.15 ± 0.13 nM, similar to that of [125I]α-Bgt in the same preparations (0.52 ± 0.16 nM). Association and dissociation kinetics were relatively rapid (kobs for association at 1 nM was 0.024 ± 0.002 min-1; koff = 0.0195 ± 0.001 min-1). The selectivity of [125I]α-conotoxin ArIB[V11L;V16A] was confirmed in autoradiographic experiments using α7-null mutant tissue: specific binding was abolished in all regions of α7-/- brains, while wild-type mice expressed high levels of labeling and low non-specific binding. [125I]α-conotoxin ArIB[V11L;V16A] should prove useful where α7 nAChRs are co-expressed with other subtypes which are also labeled by the existing ligands [125I]α-Bgt and / or [3H]MLA. Further, true equilibrium binding experiments could be performed on α7 nAChRs, something that is impossible with [125I]α-Bgt.

B. P. CALLAGHAN1, A. HAYTHORNTHWAITE1, R. J. CLARK2, D. J. CRAIK2, *D. J. ADAMS1;
α-Conotoxin Vc1.1 inhibits N-type calcium channels in rat dorsal root ganglion neurons via G protein-coupled receptor activation
1Sch. of Biomed. Sci., 2Inst. for Mol. Biosci., Univ. Queensland, Brisbane, Australia

Abstract: Regulation of voltage-gated calcium channel (VGCC) activity of dorsal root ganglion (DRG) neurons plays an important role in nociception and pain transmission. α-Conotoxin Vc1.1 is reported to have analgesic properties but the precise mechanism by which Vc1.1 relieves pain is unresolved. The effect of the synthetic peptide Vc1.1 on VGCC currents was investigated in dissociated rat DRG neurons. Application of Vc1.1 inhibited high voltage-activated (HVA) Ca2+ channel currents by 42 ± 3.3% (n = 35) in a concentration-dependent manner (IC50 = 30 nM) in >80% of neurons tested. The post-translationally modified native peptide, vc1a (1 mM), was inactive as were the α-conotoxins MII and [A10L]PnIA. In the presence of w-conotoxin CVID, Vc1.1 failed to further reduce the current amplitude, suggesting that Vc1.1 inhibits N-type VGCCs. However Vc1.1 at concentrations up to 10 µM was without effect on VGCC currents recorded from Xenopus oocytes expressing either CaV2.2 (N-type) or CaV2.1 (P/Q- type) channels. Inhibition of HVA Ca2+ channels in DRG neurons by Vc1.1 was not reversed by depolarizing prepulses but was abolished by either substitution of intracellular GTP with GDPbS (100 µM) or by pre-incubation of cells with pertussis toxin (PTX). These data indicate that Vc1.1 does not interact with VGCCs directly but inhibits CaV2.2 via a voltage-independent mechanism involving a G protein-coupled receptor of the PTX-sensitive Gi and Go classes in rat DRG neurons. Inclusion of a selective peptide inhibitor of pp60c-src tyrosine kinase also antagonized Vc1.1 inhibition of HVA Ca2+ channel currents, suggesting the involvement of src tyrosine kinase-mediated phosphorylation. Pre-incubation with a variety of selective receptor antagonists demonstrated that only the GABAB receptors antagonists, CGP 55845 (1 µM) and phaclofen (50 µM) blocked the effect of Vc1.1 on HVA Ca2+ channel currents. Taken together, these data identify CaV2.2 as a target of Vc1.1, potentially mediating its analgesic actions. We propose a novel mechanism by which α-conotoxin Vc1.1 modulates native N-type (CaV2.2) Ca2+ channel currents, namely as an agonist at G protein-coupled GABAB receptors.

Z. RADIC1, T. T. TALLEY1, C. KIM1, H. WOO KIM2, S. B. HANSEN1, R. E. HIBBS1, M. HAREL3, J. M. MCINTOSH2, B. M. OLIVERA2, *P. TAYLOR1;
Crystal structures of α-conotoxins OmIA and PeIA in complex with acetylcholine binding protein from Aplysia californica
1UC San Diego, La Jolla, CA; 2Dept. of Biol., Univ. of Utah, Salt lake City, UT; 3Weizmann Inst. of Sci., Rehovot, Israel

Abstract: Conotoxins are toxic peptides found in venoms of predatory Conus snails. α-conotoxins target nicotinic acetylcholine receptors (nAChRs) and conotoxin OmIA specifically binds to neuronal α7 and α3β2 nAChRs, while conotoxin PeIA preferentially binds to α9α10 and α7 nAChRs. The differential specificity of these alpha-conotoxins is also revealed in their interaction with acetylcholine-binding proteins (AChBPs) isolated from Aplysia californica, Lymnaea stagnalis and Bulinus trucatus. The molluskan AChBPs are homopentameric homologs of the extracellular binding domain of pentameric ligand-gated ion channel family. AChBPs most closely resemble the α-subunit of nicotinic acetylcholine receptors and in particular the homomeric α7 nicotinic receptor. We have crystallized and determined atomic 3D structures of complexes of AChBP from Aplysia californica with α-conotoxins OmIa and PeIA. The asymmetric subunits of the reported structures contain either one or two AChBP homopentamers. Toxin peptide molecules are found bound at the homopentamer subunit interface in a similar manner as previously found for structures of α-conotoxins IMI and PnIA, with ligand binding pockets in maximally open conformations with a radially extended C loop. The 3D structures solved in this study indicate that the individual binding specificity for each of the studied conotoxins is reflection of the differential peptide side-chain interactions with the ligand binding environment of the AChBP.

*L. AZAM1, C. DOWELL1, J. M. MCINTOSH1,2;
Alpha-conotoxin BuIA analogs distinguish among α6* nicotinic acetylcholine receptors
1Dept Biol, 2Psychiatry, Univ. Utah, Salt Lake City, UT

Abstract: α-conotoxins, isolated from the venom of various Conus species, are subtype selective antagonists of nicotinic acetylcholine receptors (nAChRs). The selectivity of α-conotoxins may be greatly increased by amino acid substitutions within their inter-cysteine loops (McIntosh et al., 2004). α-conotoxin BuIA, cloned from Conus purpurascens, blocks heteromeric nAChRs (except α4β2) with nanomolar potency. However, BuIA can kinetically distinguish among nAChRs that contain a β2 vs. β4 subunit, such that recovery from toxin block is much faster for β2- than β4-containing nAChRs. We have recently synthesized a number of BuIA analogs. One of these analogs selectively blocks α6/α3β4 vs. α6/α3β2β3 nAChRs. When tested against both rat and mouse nAChRs expressed in oocytes, the α-BuIA analog blocked α6/α3β4 nAChRs with an IC50 of 44.3 nM (95% CI: 38.5-51 nM) and 78.7 nM (95% CI: 58.1-106.6 nM), respectively. In contrast to BuIA, however, the analog had a much faster off-rate. Other nAChR subtypes were either not blocked or only modestly blocked by the toxin at high nanomolar to micromolar concentrations. To test the effect of this analog on native nAChR subtypes, it was used to block nicotine-stimulated [3H]norepinephrine (NE) release from postnatal (14-21 days old) mouse hippocampal synaptosomes, previously shown to be partially mediated by α6β4* nAChRs (Azam and McIntosh, 2006). At 1 mM, the peptide inhibited nicotine-evoked [3H]NE release by 33.6 ± 6.04%, similar to the portion of NE release that was shown to be mediated by α6β4* nAChR subtype. These results suggest that analogs of BuIA may be useful pharmacological tools in distinguishing between α6β2* and α6β4* nAChRs.

*A. J. HONE1,2, E. L. MEYER2,3, K. E. HILL6, J. W. ROSE6,4, N. G. CARLSON4,6,5,7, J. M. MCINTOSH2,3;
Cy3-ArIB[V11L;V16A]: a novel fluorescent α-conotoxin for the detection of α7 nicotinic acetylcholine receptors
1Neurosci., 2Psychiatry, 3Biol., 4Neurol., 5Neurobiology & Anat., Univ. Utah, Salt Lake City, UT; 6Neurovirology, 7Grecc, Vetrans Affairs Hlth. Care Ctr., Salt Lake City, UT

Abstract: α7 is one of the most widely distributed nicotinic acetylcholine receptor (nAChR) subtypes in the brain and can be found on non-neuronal tissues including macrophages and lymphocytes. In the central nervous system, α7 subunit-containing nAChRs have been shown to be involved in a variety of cellular processes including the modulation of synaptic transmission, Ca2+ signaling, and neurotransmitter release. Additionally, α7 nAChRs have been implicated in several clinical disorders including schizophrenia and Alzheimer's. Venom from marine cone snails contains small peptides usually between 13 and 30 amino acids in size that target multiple types of ion channels including nAChRs. These peptides can be labeled with fluorescent dyes to produce novel probes for receptor labeling. We recently described a set of novel, α7-selective α-conotoxins (Whiteaker PW, et al., 2007, Biochemistry, in press). We labeled one such analog with Cy3 to produce a fluorescent ligand, Cy3-ArIB[V11L;V16A], that is selective for the α7 nAChRs. When tested on cloned nAChRs expressed in Xenopus laevis oocytes Cy3-ArIB[V11L;V16A] was > 2000-fold more potent at blocking ACh-induced current in α7 than in α2β2, α3β2, α4β2, α2β4, α4β4, α6/α3β2β3, α6/α3β4, α9α10 and a1b1de nAChRs. Block of α7 nAChRs was only slowly reversible (26% recovery from 5 mM toxin after 60 min). In addition, Cy3-ArIB[V11L;V16A] was >50-fold more potent at α7 nAChRs than FITC conjugated a-bungarotoxin.

M. O. KOVALENKO1, D. J. SCHULZ2, *A. D. MCCLELLAN2;
Spinal cord injury induces changes in ion channels of reticulospinal neurons in larval lamprey
1Interdisciplinary Neurosci. Program, 2Div. Biol Sci., Univ. of Missouri, Columbia, MO

Abstract: In larval lamprey, spinal cord injury (SCI) results in significant changes in the firing properties of Müller cells, which are large identified reticulospinal (RS) neurons (McClellan et al., 2002). For example, during applied depolarizing current pulses, uninjured RS neurons fire a smooth train of action potentials (APs), while 2 weeks after SCI, axotomized neurons either fire a single short burst of APs or multiple short bursts. In addition, at 2 weeks following SCI the slow AHP (sAHP) and afterdepolarizing potential (ADP) were absent or significantly reduced and the fast AHP (fAHP) was significantly larger than in uninjured neurons. The spinal cord was hemi-transected on the right side at 10% body length (relative distance from the anterior head) so that right Müller cells were axotomized (injured) and left cells were uninjured (n=51 animals). First, for uninjured RS neurons, blocking HVA calcium channels (N- and P/Q-type) with 2 mM w-conotoxin MVIIC significantly reduced the sAHP to 2.5%±5.8% of control values (N=11 neurons) and produced changes in firing patterns in response to applied depolarizing current pulses that mimicked some of the effects of SCI. The sAHP also was significantly reduced to 5.6%±9.7% of control values by blocking only N-type calcium channels with 2 mM w-conotoxin GVIA (N=11 neurons). Furthermore, blocking SKKCa channels with 20 mM apamin significantly reduced the sAHP to 8.1%±14.1% of control values (N=11 neurons). Second, computer modeling indicated that the AP and firing patterns of axotomized RS neurons could be mimicked by substantially reducing HVA calcium channels and SKKCa channel conductances compared to those in models of uninjured neurons, as well as some other minor changes in model parameters. Third, at relatively long recovery times (12-16 weeks) axotomized RS neurons often displayed firing patterns and sAHP that were similar to those of uninjured neurons (N=32). Fourth, molecular biology studies demonstrated that expression of HVA calcium and SKKCa channels in axotomized RS neurons was significantly down-regulated at short recovery times (n=9 animals; 1 wk) and was restored at long recovery times (n=5 animals; 11-17 wks). We previously demonstrated that calcium influx in RS neurons in culture results in inhibition of neurite outgrowth (Ryan et al., 2004, 2007). Together, these results suggest that following SCI, axotomized RS neurons down-regulate calcium channels to reduce calcium influx and maintain intracellular calcium levels in a range that is permissive for axonal regeneration.

*E. GRAZZINI, H. VU, S. ZICHA, M. DUCHESNE, M. VALIQUETTE, S. AHMAD, P. LEMBO;
Functional and pharmacological characterization of a voltage gated calcium channel using the FLIPR and PatchXpress
Astrazeneca, Ville St Laurent, PQ, Canada

Abstract: An important component of studying voltage- and ligand-gated ion channels is the ability to accurately measure the activity of compounds on ion channel function. The gold standard assay for high-quality measurements of ion channel function is the patch-clamp technique. However, even with conventional patch-clamp, accurate determination of effective concentrations (EC50/IC50) is not necessarily straightforward and is time consuming. One example of the difficulty encountered is found in the wide range of IC50 values reported in the literature for compounds that block the hERG potassium channel. IC50 values for individual compounds have been reported that span two orders of magnitude. Patch-clamp and FLIPR techniques have been used to study the biophysical and pharmacological properties of the rat orthologue of the voltage gated calcium channel Cav2.2. Using FLIPR, we were able to demonstrate that rat Cav2.2 cell lines show a saturable and dose-dependent calcium response to KCl stimulation. The well-known channel blocker w-conotoxin MVIIA (200nM) as well as the novel AstraZeneca compound AZ1 (5mM), showed 95% and 82% inhibition (n=5) respectively of the KCl induced calcium response. Using this method, we demonstrate that FLIPR can be used as a screening method to identify Cav2.2 channel blockers including conotoxins, as well as use-dependent antagonists including AZ1. The PatchXpress 7000A, an automated patch clamp system (Molecular Devices), was used to develop a Cav2.2 assay that can examine channel biophysics and pharmacology. Cav2.2 kinetics, current amplitude and pharmacological profile using reference compounds as well as AZ1 were compared using the PatchXpress and conventional patch-clamp and comparable results were obtained with both systems. Because Cav2.2 antagonists are likely to be more effective during the enhanced activation patterns in pain syndromes (high-frequency), AZ1’s mechanism of action has been confirmed with a protocol designed to assess the use-dependency at different frequencies (0.1, 0.2, 0.5, 1 Hz). The results presented herein demonstrate that the data obtained with the PatchXpress is comparable in quality to that of conventional patch-clamp with the added benefits of higher throughput and accurate measurement of channel kinetics.

*K. YANG1, R. J. LUKAS2, J. WU1;
Functional a-6 nicotinic acetylcholine receptors located on pre-synaptic, GABAergic boutons in rat midbrain dopaminergic neurons participate in cholinergic modulation of GABA release
1Neurol, Barrow Neurologicial Inst., Phoenix, AZ; 2Div. of Neurobiology,Barrow Neurological Inst., St. Joseph’s Hospital and Medical Center, Phoenix, AZ

Abstract: The ventral tegmental area (VTA) plays a central role in reward, motivation and drug addiction. Nicotinic acetylcholine receptor (nAChR) α6 subunits are expressed in the VTA at high levels and are thought to constitute α6*-nAChR that mediate cholinergic effects on dopaminergic (DA) function. However, direct evidence is lacking for functional α6*-nAChRs and their roles in the VTA. Here, we test the hypothesis that functional α6*-nAChRs are located on pre-synaptic, GABAergic boutons of VTA DA neurons, where they mediate cholinergic modulation of GABA release. Perforated patch-clamp whole-cell recording from mechanically (no enzyme) dissociated neurons (maintaining pre-synaptic boutons) from 8-14 postnatal day rats revealed spontaneous inward currents at a holding potential of -60 mV that were abolished by the GABAA receptor antagonist, bicuculline methiodide (BMI). Bath application of acetylcholine (ACh,1 mM plus atropine 500 nM) enhanced the frequency of these apparent, spontaneous inhibitory postsynaptic currents (sIPSCs), but selective activation of α4β2- (RJR-2403 100 mM) or α7- (choline 10 mM) nAChRs failed to increase sIPSC frequency. Interestingly, the ACh-induced increase of sIPSC was abolished by a relatively selective, α6*-nAChR antagonist, α-conotoxin MII (1-10 nM), but not by antagonists selective for α4β2- (1 mM dihydro-beta-erythroidine) or α7- (10 nM methyllycaconitine) nAChR. This suggested that ACh activates functional α6-containing nAChRs located on pre-synaptic, GABAergic boutons contacting VTA DA neurons and increases GABA release. A smoking-relevant concentration (500 nM) of nicotine did not acutely alter sIPSC frequency, but a 4 min pretreatment desensitized α6*-nAChRs and abolished ACh-induced increases in sIPSCs. Collectively, our results demonstrate, for the first time, that functional α6*-nAChRs are expressed on GABAergic terminals/boutons in the VTA and may play a critical role in mediating cholinergic modulation of GABA release. Their desensitization during chronic nicotine exposure may contribute to disinhibition of VTA DA neuronal activity and DA release, perhaps helping to explain nicotine-induced reinforcement.

*T. MCCLURE-BEGLEY, S. R. GRADY, P. WHITEAKER, M. J. MARKS, A. C. COLLINS;
Modulation of nachr-evoked neurotransmitter release by presynaptic gabab receptors
Inst. Behav Genet., Univ. Colorado, Boulder, CO

Abstract: The nature of GABAB receptor-mediated inhibition of 3H-GABA and 3H-Dopamine (DA) release stimulated by nicotinic receptor activation from striatal synaptosomes was evaluated in an attempt to elucidate the interactions of presynaptic inhibitory and excitatory receptors that influence neurotransmitter exocytosis. The selective GABAB agonist (R)-baclofen (0.3-300mM) dose-dependently inhibited the acetylcholine (ACh) stimulated release of 3H-GABA from striatal synaptosomes, with maximum inhibition approaching 100%. The dose-response relationship for (R)-baclofen inhibition of GABA release is best fit to a single-site model, suggesting an effect mediated by a single receptor. Potassium depolarization-evoked release of both 3H-GABA and 3H-DA were partially decreased following a 30 second exposure to 100mM (R)-baclofen, suggesting that GABAB receptors are present on at least a proportion of both GABAergic as well as DAergic nerve terminals in the striatum. However, treatment of striatal synaptosomes with 100mM (R)-baclofen for 30 seconds prior to stimulation with ACh caused a marked decrease in 3H-GABA release, whereas no significant depression of 3H-DA release was observed. These experiments were conducted using physiological buffer supplemented with 10mM CsCl to prevent the activation of inwardly-rectifying potassium channels (a known target of postsynaptic GABAB modulation). This result suggested that under identical conditions, the nature of GABAB receptor-mediated inhibition of neurotransmitter release evoked by ACh differs according to cell type. Experiments to elucidate the mechanism implicated in this effect are currently ongoing. Presynaptic GABAB receptors are often coupled to N-type high-voltage gated calcium channels (CaV2.2), and inhibit neurotransmitter release by interacting with the pore-forming subunit of the calcium channel. This would imply that GABA release stimulated by ACh from striatal synaptosomes is entirely dependent on CaV2.2 activation. However, 3H-GABA release evoked by both potassium and ACh is completely insensitive to the CaV2.2 antagonist w-conotoxin GVIA, suggesting a novel mechanism is responsible for (R)-baclofen-induced inhibition of ACh-evoked 3H-GABA release from striatal GABAergic nerve terminals.

*C. STOKES1, L. P. DWOSKIN2, P. A. CROOKS2, L. B. JACOBS1, J. M. MCINTOSH3, R. L. PAPKE1;
The identification of selective competitive and noncompetitive nAChR antagonists using alphα6 nicotinic acetylcholine receptor chimeras
1Pharmacol. & Therapeut., Univ. of Florida, Gainesville, FL; 2Univ. of Kentucky, Lexington, KY; 3Univ. of Utah, Salt Lake City, UT

Abstract: Heterologous expression systems have increased the feasibility of developing selective ligands to target nicotinic acetylcholine receptor (nAChR) subtypes. However, some important native receptor subtypes are difficult to express in systems such as the Xenopus oocyte, which presents a challenge to this approach. The α6 subunit, a component in nAChRs that mediates some of the reinforcing effects of nicotine self-administration, is one such problematic subunit. Certain aspects of α6-containing receptor pharmacology have been studied by using chimeric subunits containing the α6 ligand-binding domain. However, some nAChR antagonists such as mecamylamine, which may be useful for treating nicotine dependence, act within the channel pore rather than at the ACh binding site. Therefore, we generated an α6 chimera (α4/6) that has the transmembrane and intracellular domains of α6 and the extracellular domain of α4. We show that, as expected, receptors containing the α4/6 chimera are insensitive to the α-conotoxin PIA, a selective antagonist for reciprocal chimeras with the α6 extracellular domain. We examined the pharmacological properties of α4/6-containing receptors and other important nAChR subtypes, including α7, α4β2, α4β4, α3β4, α3β2, and α3β2β3, as well as receptors containing both the α6 extracellular chimeras α6/3 and α6/4. Our data show that the presence or absence of the β4 subunit is an important factor for sensitivity to the ganglionic blocker mecamylamine, and we provide additional support for dihydro-b-erythroidine being most effective on subtypes containing α4 subunits. We evaluated the activity and selectivity of a series of novel quaternary ammonium compounds that have high potency for partial blockade of nicotine-evoked dopamine release from striatal slices and have identified compounds such as GZ-580A, which, at a probe concentration of 1 µM, produced significantly more inhibition of receptors containing the α6 intracellular and transmembrane domains than receptors containing the corresponding domains of α4. Due to the role of α6-containing receptors in dopamine-mediated nicotine reward, such α6-selective noncompetitive antagonists may be useful smoking cessation therapies.

B. FIEDLER1, M. ZHANG1, L. AZAM1, G. BULAJ2, B. M. OLIVERA1, O. BUCZEK1, *D. YOSHIKAMI1;
Excitatory iota-conotoxin RXIA modulates the voltage sensitivity of activation of NaV1.2, 1.6, and 1.7
1Dept Biol, 2Dept Med. Chem, Univ. Utah, Salt Lake City, UT

Abstract: Iota-toxins in the I1 superfamily of conopeptides induce repetitive action potentials in frog motor axons (Jiminez et al., 2003 J. Neurochem. 85, 610; Buczek et al., 2005 J. Biol. Chem. 280, 4247, & FEBS J. 272, 4178). We recently found that i-RXIA, a founding member of the I1 superfamily, modulates mouse NaV1.6 by left-shifting its voltage dependence of activation (Buczek et al., 2007 Am. Peptide Soc. Symp. abstract #417). Here, we examined i-RXIA’s selectivity by testing it on cloned NaV1.1 through 1.7 co-expressed with b1 in Xenopus oocytes. A convenient measure of i-RXIA-activity was the increase in peak INa to a fixed test voltage step to -30 mV (DINa-30). These measurements, as well as leftward shifts in the V1/2 of activation, indicated that of the seven NaV1 isoforms tested, only NaV1.2, 1.6, and 1.7 appeared to be sensitive to i-RXIA. Unlike b-scorpion toxins, the effects of i-RXIA seemed insensitive to depolarizing conditioning pulses. The affinity of i-RXIA for NaV1.6, determined from the kinetics of DINa-30 following exposure to various concentrations of i-RXIA (which were well fit by single-exponential curves), indicated a Kd of 2.9 mM, close to the EC50 of 1.8 mM obtained from the plot of steady-state DINa-30 versus i-RXIA concentration. The Kd and EC50 for NaV1.2 were likewise determined and found to be similar, 13.8 and 17.8 mM, respectively. The effect of i-RXIA on NaV1.7 was small, with an estimated EC50 > 20 mM. An intriguing structural feature of i-RXIA is that Phe 44 (the 3rd residue from the C-terminus of this 46 AA peptide) is post-translationally epimerized to D-Phe. To examine the functional role of this unusual modification, we synthesized the i-RXIA analog where residue 44 was an L-Phe (i.e., i-RXIA[L-Phe44]) and tested it against NaV1.6. Its Kd and EC50 were similar (5.5 and 5.4, respectively), about double those of i-RXIA. The kon of i-RXIA[L-Phe44] was about the same as that of i-RXIA; however, its koff was two-fold faster, which accounts for the difference in the Kd between the two peptides. Of particular interest is the observation that at saturating concentrations of peptide, the DINa-30 induced by i-RXIA was about twice that by i-RXIA[L-Phe44]; i.e., when the i-toxin binding site on the channel was fully occupied, the functional effect differed depending on the bound peptide. This indicates that the binding and the efficacy of these peptides may be uncoupled. Finally, when the sciatic nerve of the mouse was exposed to 50 nM i-RXIA, extracellular recordings revealed that repetitive action potentials were induced in both A- and C-fibers, consistent with the presence of NaV1.6 in both myelinated and unmyelinated PNS axons.

B. HRUSKOVA1, J. TROJANOVA1, A. KLUG2, M. GASSMANN3, B. BETTLER3, *H. R. BRENNER3, R. TURECEK1;
Modulation of excitatory synaptic transmission by GABAB receptors and SK channels in the medial nucleus of trapezoid body
1Inst. of Exptl. Medicine, AS CR, Prague, Czech Republic; 2Dept of Neurobiology, Ludwig-Maximilians-University, Munich, Germany; 3Dept of Physiology, Univ. of Basel, Basel, Switzerland

Abstract: Calcium-dependent potassium channels (KCa) are activated by low concentrations of Ca2+ and hyperpolarize a wide range of neuronal cells. GABAB receptors are G-protein coupled receptors that are inhibitory to voltage-gated Ca2+ channels (VGCC). The tight coupling between intracellular Ca2+ and the activity of KCa suggests that the inhibition of VGCC by GABAB receptors could subsequently result in a reduction of KCa currents and KCa-induced hyperpolarizations. Principal cells (PCs) of the medial nucleus of the trapezoid body (MNTB) accurately convert excitatory signals to inhibitory signals and the precise action of the MNTB synapses is important for sound source localization in mammals. The firing pattern of PCs is profoundly affected by the afterhyperpolarization phase (AHP) following a postsynaptic action potential (AP). The aim of this study was to investigate the indirect modulatory effects of GABAB receptors on KCa and KCa-mediated AHP in PCs using mouse MNTB slices. APs were evoked by current step injections into PCs and recorded by the patch clamp technique. The APs were followed by AHP consisting of a fast and a slow component. The slow component was sensitive to apamin, bicuculline and intracellular BAPTA and was enhanced after a high frequency train of action potentials. This indicated that it was mediated by small conductance KCa channels (SK). The MNTB slices were intensively stained with antibodies raised against SK1 and SK2 (but not against SK3), suggesting that these were the main subtypes of SK channels in MNTB PCs. Baclofen, a GABAB agonist, reduced the slow component leaving the fast one unaffected. The slow component was also blocked by Cd2+, w-conotoxin GVIA and w-agatoxin IVA, and it was insensitive to nimodipin. This indicates that the activity of KCa and slow AHP was induced by the opening of N- and P/Q-type VGCC. The baclofen-induced modulation of AHP was absent in mice lacking the GABAB1b subunit isoform. Thus, the GABAB1b appeared to be the dominant isoform of GABAB1 forming postsynaptic receptors indirectly coupled to SK channels. To test the effects of GABAB-mediated modulation of AHP on the ability of PCs to follow high frequency synaptic stimuli we have used the dynamic clamp technique. Trains of 20 current injection waveforms delivered at 100 - 500 Hz to PCs evoked both action potentials and frequent failures. Baclofen significantly reduced the number of failures in a frequency dependent manner indicating that GABAB modulate the firing of PCs by inhibiting KCa-mediated AHP. Our results reveal interactions between postsynaptic VGCC, KCa and GABAB that ensure the reliability of excitatory transmission at an auditory synaptic relay station.

A. R. HANSLER, D. T. KOZLOSKY, *L. B. FRENCH;
The effects of Conus textile venom on two potassium channels, hSlo 1.1 and lShal
Dept Biol, Allegheny Col., Meadville, PA

Abstract: Small peptides in Conus snail venoms, known as conotoxins, cause excitotoxic shock and prevent neuromuscular synaptic transmission by disrupting the activity of specific ion channels. Due to their unmatched binding specificity for their targets, conotoxins can be used to characterize ligand- and voltage-gated ion channels. Conotoxins have already been shown to be useful tools for both research and clinical applications. hSlo 1.1 is a human calcium-activated K+ channel found in vascular smooth muscle. lShal is a voltage-gated K+ channel found in the stomatogastric nervous system of Panulirus interruptus. Both channels play critical roles in regulating the excitability of cells involved in rhythmic motor outputs. In this study, Xenopus oocytes were injected with hSlo 1.1 or lShal RNA. Currents were characterized using two-electrode voltage clamp, and then 0.5 mg Conus textile venom in physiological saline was bath-applied to the oocytes in a 0.3 ml recording volume. The venom significantly increased macroscopic K+ currents in approximately 80% of the channel-expressing oocytes tested, while having no effect on oocytes injected with water. Although the molecular mechanisms behind these phenomena are unknown, the effects appear to be voltage-dependent. It is hypothesized that two different conotoxins are targeting hSlo and lShal. Further work is necessary to identify the venom components affecting the channels, which could lead to the development of valuable research tools for the study of vascular smooth muscle regulation and invertebrate neural networks.