Layla Azam

Co-expression of α7 and β2 nicotinic acetylcholine receptor subunit mRNAs within rat brain cholinergic neurons

Nicotine enhances cognitive and attentional processes through stimulation of the basal forebrain cholinergic system. Although muscarinic cholinergic autoreceptors have been well characterized, pharmacological characterization of nicotinic autoreceptors has proven more difficult. The present study used double-labeling in situ hybridization to determine expression of nicotinic acetylcholine receptor (nAChR) subunit mRNAs within basal forebrain cholinergic neurons in order to gain information about possible nAChR autoreceptor properties. Cholinergic cells of the mesopontine tegmentum and striatal interneurons were also examined, as were septohippocampal GABAergic neurons that interact with cholinergic neurons to regulate hippocampal activity. alpha7 and beta2 nAChR mRNAs were found to be co-expressed in almost all cholinergic cells and in the majority of GABAergic neurons examined. alpha4 nAChR mRNA expression was restricted to cholinergic cells of the nucleus basalis magnocellularis, and to non-cholinergic cells of the medial septum and mesopontine tegmentum. These data suggest possible regional differences in the pharmacological properties of nicotinic autoreceptors on cholinergic cells. Whereas most cholinergic cells express rapidly desensitizing alpha7 homomers or alpha7beta2 heteromers, cortical projection neurons may also express a pharmacologically distinct alpha4beta2 nAChR subtype. There may also be differential nAChR regulation of cholinergic and non-cholinergic cells within the mesopontine tegmentum that are implicated in acquisition of nicotine self-administration.

Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors

AbstractCysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the α-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain α-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinsons disease and nociception. Structure/function characterization of α-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of α-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of α-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.

Developmental regulation of nicotinic acetylcholine receptors within midbrain dopamine neurons.

We have combined anatomical and functional methodologies to provide a comprehensive analysis of the properties of nicotinic acetylcholine receptors (nAChRs) on developing dopamine (DA) neurons of Sprague-Dawley rats. Double-labeling in situ hybridization was used to examine the expression of nAChR subunit mRNAs within developing midbrain DA neurons. As brain maturation progressed there was a change in the pattern of subunit mRNA expression within DA neurons, such that alpha3 and alpha4 subunits declined and alpha6 mRNA increased. Although there were strong similarities in subunit mRNA expression in substantia nigra (SNc) and ventral tegmental area (VTA), there was higher expression of alpha4 mRNA in SNc than VTA at gestational day (G) 15, and of alpha5, alpha6 and beta3 mRNAs during postnatal development. Using a superfusion neurotransmitter release paradigm to functionally characterize nicotine-stimulated release of [(3)H]DA from striatal slices, the properties of the nAChRs on DA terminals were also found to change with age. Functional nAChRs were detected on striatal terminals at G18. There was a decrease in maximal release in the first postnatal week, followed by an increase in nicotine efficacy and potency during the second and third postnatal weeks. In the transition from adolescence (postnatal days (P) 30 and 40) to adulthood, there was a complex pattern of functional maturation of nAChRs in ventral, but not dorsal, striatum. In males, but not females, there were significant changes in both nicotine potency and efficacy during this developmental period. These findings suggest that nAChRs may play critical functional roles throughout DA neuronal maturation.

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. μ-Conotoxin KIIIA is representative of μ-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ∼20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both NaV1.2 and NaV1.6 were strongly blocked; within experimental wash times of 40–60 min, block was reversed very little for NaV1.2 and only partially for NaV1.6. Other isoforms were blocked reversibly: NaV1.3 (IC50 8 μm), NaV1.5 (IC50 284 μm), and NaV1.4 (IC50 80 nm). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4; replacement of Trp-8 resulted in reversible block of NaV1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of NaV1.2 and that further engineering of μ-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve

Voltage-gated sodium channels (VGSCs) are important for action potentials. There are seven major isoforms of the pore-forming and gate-bearing α-subunit (NaV1) of VGSCs in mammalian neurons, and a given neuron can express more than one isoform. Five of the neuronal isoforms, NaV1.1, 1.2, 1.3, 1.6, and 1.7, are exquisitely sensitive to tetrodotoxin (TTX), and a functional differentiation of these presents a serious challenge. Here, we examined a panel of 11 μ-conopeptides for their ability to block rodent NaV1.1 through 1.8 expressed in Xenopus oocytes. Although none blocked NaV1.8, a TTX-resistant isoform, the resulting “activity matrix” revealed that the panel could readily discriminate between the members of all pair-wise combinations of the tested isoforms. To examine the identities of endogenous VGSCs, a subset of the panel was tested on A- and C-compound action potentials recorded from isolated preparations of rat sciatic nerve. The results show that the major subtypes in the corresponding A- and C-fibers were NaV1.6 and 1.7, respectively. Ruled out as major players in both fiber types were NaV1.1, 1.2, and 1.3. These results are consistent with immunohistochemical findings of others. To our awareness this is the first report describing a qualitative pharmacological survey of TTX-sensitive NaV1 isoforms responsible for propagating action potentials in peripheral nerve. The panel of μ-conopeptides should be useful in identifying the functional contributions of NaV1 isoforms in other preparations.

Structure/function characterization of -conotoxin kiiia, an analgesic, nearly irreversible blocker of neuronal mammalian sodium channels

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. μ-Conotoxin KIIIA is representative of μ-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ∼20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both NaV1.2 and NaV1.6 were strongly blocked; within experimental wash times of 40–60 min, block was reversed very little for NaV1.2 and only partially for NaV1.6. Other isoforms were blocked reversibly: NaV1.3 (IC50 8 μm), NaV1.5 (IC50 284 μm), and NaV1.4 (IC50 80 nm). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4; replacement of Trp-8 resulted in reversible block of NaV1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of NaV1.2 and that further engineering of μ-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

Characterization of Nicotinic Acetylcholine Receptors that Modulate Nicotine-evoked [3H]Norepinephrine Release from Mouse Hippocampal Synaptosomes

Nicotines modulation of hippocampal noradrenergic neurotransmission may contribute to its mnemonic properties, but the nicotinic acetylcholine receptor (nAChR) subtypes that modulate terminal release of norepinephrine are unknown. In the present study, we used a number of subtype-selective α-conotoxins in combination with nicotinic receptor subunit-deficient mice to characterize nAChRs that modulate [3H]nore-pinephrine release from synaptosomes. The results indicate that at least two populations of nAChRs contribute to this release: a novel α6(α4)β2β3β4 subtype and an α6(α4)β2β3 subtype. These are distinct from subtypes that modulate synaptosomal norepinephrine release in the rat hippocampus in which an α6/β2 and/or α6/β4 ligand binding interface is not present. Whereas α-conotoxin MII fully inhibits nicotine-evoked [3H]norepinephrine release in mouse, it is ineffective in blocking [3H]norepinephrine release in rat. Block of [3H]norepinephrine release by α-conotoxin BuIA, a toxin that kinetically distinguishes between β2- and β4-containing nAChRs, was partially reversible in mouse but irreversible in rat. This indicates that in contrast to rat, mouse nAChRs are made of both β4 and non-β4-containing populations. Results from β2 and β4 null mutant mice confirmed this conclusion, indicating the presence of the β2 subunit in all nAChRs and the presence of the β4 subunit in a subpopulation of nAChRs. In addition, both α4 and β3 subunits are essential for the formation of functional nAChRs on mouse noradrenergic terminals. Cytisine, a ligand formerly believed to be β4-selective, was a highly effective agonist for α6β2-containing nAChRs. The sum of these results suggests a possible novel nAChR subtype that modulates nor-adrenergic neurotransmission within the mouse hippocampus.

Structure, dynamics and selectivity of the sodium channel blocker mu-conotoxin SIIIA

mu-SIIIA, a novel mu-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-resistant sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remains unknown. We have determined the structure of mu-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, mu-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11-16, while residues 3-5 in the N-terminal region form a helix-like turn resembling 3 10-helix. The Trp12 and His16 side chains are close together, as in the related conotoxin mu-SmIIIA, but Asn2 is more distant. Dynamics measurements show that the N-terminus and Ser9 have larger-magnitude motions on the subnanosecond time scale, while the C-terminus is more rigid. Cys4, Trp12, and Cys13 undergo significant conformational exchange on microsecond to millisecond time scales. mu-SIIIA is a potent, nearly irreversible blocker of Na V1.2 but also blocks Na V1.4 and Na V1.6 with submicromolar potency. The selectivity profile of mu-SIIIA, including poor activity against the cardiac sodium channel, Na V1.5, is similar to that of the closely related mu-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal Na V1.2. The structural and functional characterization described in this paper of an analgesic mu-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.

α-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between α6β4 and α6β2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release

α6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of α6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. α-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of α-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyxproline increased the IC(50) by 2800-fold at α6/α3β2β3 but only by 6-fold at α6/α3β4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepinephrine release. Release was abolished in α6-null mutant mice. α-Conotoxin BuIA[T5A;P6O] partially blocked norepinephrine release in wild-type controls but failed to block release in β4(-/-) mice. In contrast, BuIA[T5A;P6O] failed to block dopamine release in the wild-type striatum known to contain α6β2* nAChRs. BuIA[T5A;P6O] is a novel ligand for distinguishing between closely related α6* nAChRs; α6β4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum.

Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA

J. Neurochem. (2012) 122, 1137–1144.