Ron C. Hogg

Crystal Structure of Nicotinic Acetylcholine Receptor Homolog Achbp in Complex with an Alpha-Conotoxin Pnia Variant

Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup α-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 Å of α-Ctx PnIA (A10L D14K), a potent blocker of the α7-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. α-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.

A New Level of Conotoxin Diversity, a Non-native Disulfide Bond Connectivity in alpha -Conotoxin AuIB Reduces Structural Definition but Increases Biological Activity.

α-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2–8 and 3–15) and the ribbon isomer having the disulfide connectivity Cys(2–15 and 3–8). The solution structures of the peptides were determined by NMR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Single Amino Acid Substitutions in α-Conotoxin PnIA Shift Selectivity for Subtypes of the Mammalian Neuronal Nicotinic Acetylcholine Receptor

The α-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication. In this study, the native α-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the ACh-induced current in all rat parasympathetic neurons examined, with IC50 values of 14 and 33 nm, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified α-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC50 value of 375 nm and a maximally effective concentration caused 91% block. [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in ∼80% of neurons, with an IC50 value of 1.4 nm and 46% maximal block of the total current. The residual current was not inhibited further by α-bungarotoxin, but was further reduced by the α-conotoxins PnIA or PnIB, and by mecamylamine. 1H NMR studies indicate that PnIA, PnIB, and the analogues, [A10L]PnIA and [N11S]PnIA, have identical backbone structures. We propose that positions 10 and 11 of PnIA and PnIB influence potency and determine selectivity among α7 and other nAChR subtypes, including α3β2 and α3β4. Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conopeptides.

Functional maturation of isolated neural progenitor cells from the adult rat hippocampus

Although neural progenitor cells (NPCs) may provide a source of new neurons to alleviate neural trauma, little is known about their electrical properties as they differentiate. We have previously shown that single NPCs from the adult rat hippocampus can be cloned in the presence of heparan sulphate chains purified from the hippocampus, and that these cells can be pushed into a proliferative phenotype with the mitogen FGF2 [Chipperfield, H., Bedi, K.S., Cool, S.M. & Nurcombe, V. (2002) Int. J. Dev. Biol., 46, 661–670]. In this study, the active and passive electrical properties of both undifferentiated and differentiated adult hippocampal NPCs, from 0 to 12 days in vitro as single‐cell preparations, were investigated. Sparsely plated, undifferentiated NPCs had a resting membrane potential of ≈ −90 mV and were electrically inexcitable. In > 70%, ATP and benzoylbenzoyl‐ATP evoked an inward current and membrane depolarization, whereas acetylcholine, noradrenaline, glutamate and GABA had no detectable effect. In Fura‐2‐loaded undifferentiated NPCs, ATP and benzoylbenzoyl‐ATP evoked a transient increase in the intracellular free Ca2+ concentration, which was dependent on extracellular Ca2+ and was inhibited reversibly by pyridoxalphosphate‐6‐azophenyl‐2′‐4′‐disulphonic acid (PPADS), a P2 receptor antagonist. After differentiation, NPC‐derived neurons became electrically excitable, expressing voltage‐dependent TTX‐sensitive Na+ channels, low‐ and high‐voltage‐activated Ca2+ channels and delayed‐rectifier K+ channels. Differentiated cells also possessed functional glutamate, GABA, glycine and purinergic (P2X) receptors. Appearance of voltage‐dependent and ligand‐gated ion channels appears to be an important early step in the differentiation of NPCs.

Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins A PARADIGM FOR BIOLOGICAL ACTIVITY DIVERSIFICATION

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of α-cobratoxin (a long-chain α-neurotoxin) and heterodimers formed by α-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26–30 in polypeptide loop II of α-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the α-cobratoxin capacity to compete with α-bungarotoxin for binding to Torpedo and α7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that α-cobratoxin dimer not only interacts with α7 nAChR but, in contrast to α-cobratoxin monomer, also blocks α3β2 nAChR. In the latter activity it resembles κ-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric α3β2 nAChRs.

Reactive oxygen species modulate neuronal excitability in rat intrinsic cardiac ganglia

Reactive oxygen species (ROS) are produced as by-products of oxidative metabolism and occur in the heart during ischemia and coronary artery reperfusion. The effects of ROS on the electrophysiological properties of intracardiac neurons were investigated in the intracardiac ganglion (ICG) plexus in situ and in dissociated neurons from neonatal and adult rat hearts using the whole-cell patch clamp recording configuration. Bath application of ROS donors, hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (t-BHP) hyperpolarized, and increased the action potential duration of both neonatal and adult ICG neurons. This action was also recorded in ICG neurons in an adult in situ ganglion preparation. H2O2 and t-BHP also inhibited voltage-gated calcium channel (VGCC) currents and shifted the current–voltage (I–V) relationship to more hyperpolarized potentials. In contrast, H2O2 increased the amplitude of the delayed rectifier K+ current in neonatal ICG neurons. In neonatal ICG neurons, bath application of either superoxide dismutase (SOD) or catalase, scavengers of ROS, prior to H2O2 attenuated the hyperpolarizing shift but not the inhibition of VGCC by H2O2. In contrast, in adult ICG neurons, application of SOD alone had no effect upon either VGCC current amplitude or the I–V relationship, whereas application of SOD prior to H2O2 exposure abolished both the H2O2-mediated hyperpolarizing shift and inhibition. These data indicate that ROS alter depolarization-activated Ca2+ and K+ conductances which underlie neuronal excitability of ICG neurons. This affects action potential duration and therefore probably modifies autonomic control of the heart during ischemia/reperfusion.

Ciguatoxin (CTX-1) modulates single tetrodotoxin-sensitive sodium channels in rat parasympathetic neurones.

The actions of the marine neurotoxin, ciguatoxin-1 (CTX-1), were investigated in isolated parasympathetic neurones from neonatal rat intracardiac ganglia using patch-clamp recording techniques. Under current clamp conditions, bath application of 1-10 nM CTX-1 caused gradual membrane depolarization and tonic action potential firing. Action potential firing ceased with depolarization beyond approximately -35 mV and application of 300 nM tetrodotoxin (TTX) repolarized the cell to its control resting potential. In cell-attached membrane patches, 1-10 nM CTX-1 in the patch pipette markedly increased the open probability of single TTX-sensitive Na+ channels in response to depolarizing voltage steps but did not alter the unitary conductance (10 pS) or reversal potential. Under steady-state conditions, CTX-1 caused spontaneous opening of single Na+ channels which did not inactivate at hyperpolarized membrane potentials. CTX-1 increases neuronal excitability by shifting the voltage of activation of TTX-sensitive Na+ channels to more negative potentials.

Ciguatoxin-induced oscillations in membrane potential and action potential firing in rat parasympathetic neurons.

The actions of ciguatoxins from the Pacific (P‐CTX‐1) and Caribbean (C‐CTX‐1) regions were investigated in isolated parasympathetic neurons from rat intracardiac ganglia using patch‐clamp recording techniques. Under current‐clamp conditions, bath application of P‐CTX‐1 (1–10 nm) or C‐CTX‐1 (10–30 nm) caused a gradual depolarization that was accompanied by oscillation of the membrane potential leading to tonic action potential firing. Membrane potential oscillations were observed between −45 and −60 mV and had an amplitude of 10–20 mV and a mean frequency of 10 Hz. Oscillation frequency was temperature‐dependent with a Q10 of 2.0. Membrane oscillations were temporarily inhibited by hyperpolarizing current pulses and potentiated by weak depolarizing current pulses. The amplitude of oscillations was reduced upon lowering the external Na+ concentration and inhibited by tetrodotoxin (TTX), tetracaine or Zn2+. Tetraethylammonium, 4‐aminopyridine, Cs+, Cd2+, Ba2+, 1,4,4′‐diothiocyanato‐2,2′‐stilbenedisulphonic acid (DIDS) and ouabain had no effect on the CTX‐1‐induced membrane depolarization and oscillations. Brevetoxin (PbTx‐3, 100 nm), in contrast to CTX‐1, caused a membrane depolarization that was not associated with oscillation of the membrane potential. Under voltage‐clamp conditions, P‐CTX‐1 inhibited the peak amplitude of the voltage‐dependent Na+ current and shifted the activation curve to more negative potentials, but membrane oscillations were not seen in this configuration. These results suggest that ciguatoxins cause oscillation of the membrane potential in mammalian autonomic neurons by modifying the activation and inactivation properties of a population of TTX‐sensitive Na+ channels.

Bacterial Expression, NMR, and Electrophysiology Analysis of Chimeric Short/Long-chain α-Neurotoxins Acting on Neuronal Nicotinic Receptors

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain α-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain α-neurotoxins block both muscle-type and α7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of α- and κ-neurotoxins is essential for their action on the α7 and α3β2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain α-neurotoxins and κ-neurotoxins. The correct folding and structural stability for both chimeras were shown by 1H and 1H-15N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockα7 nAChRs with similar potency (IC50 6.1 and 34 nm, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain α-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for α7 nAChR (IC50 126 nm) but did not convey activity at α3β2 nAChRs. Docking of both chimeras toα7 andα3β2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of κ-neurotoxins underlie their selectivity for α3β2 nAChRs.

Hydrophobic residues at position 10 of α-conotoxin PnIA influence subtype selectivity between α7 and α3β2 neuronal nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimers disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3β2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3β2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki=44nM) though selectivity reverted to α3β2 (IC50=0.7nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3β2 nAChRs via different molecular determinants.