Christoph Methfessel

The Novel 7 Nicotinic Acetylcholine Receptor Agonist N-((3R)-1-Azabicyclo(2.2.2)oct-3-yl)-7-(2-(methoxy)phenyl)-1- benzofuran-2-carboxamide Improves Working and Recognition Memory in Rodents

The relative contribution of α4β2, α7 and other nicotinic acetylcholine receptor (nAChR) subtypes to the memory enhancing versus the addictive effects of nicotine is the subject of ongoing debate. In the present study, we characterized the pharmacological and behavioral properties of the α7 nAChR agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide (ABBF). ABBF bound to α7 nAChR in rat brain membranes (Ki = 62 nM) and to recombinant human 5-hydroxytryptamine (5-HT)3 receptors (Ki = 60 nM). ABBF was a potent agonist at the recombinant rat and human α7 nAChR expressed in Xenopus oocytes, but it did not show agonist activity at other nAChR subtypes. ABBF acted as an antagonist of the 5-HT3 receptor and α3β4, α4β2, and muscle nAChRs (at higher concentrations). ABBF improved social recognition memory in rats (0.3–1 mg/kg p.o.). This improvement was blocked by intracerebroventricular administration of the α7 nAChR antagonist methyllycaconitine at 10 μg, indicating that it is mediated by α7 nAChR agonism. In addition, ABBF improved working memory of aged rats in a water maze repeated acquisition paradigm (1 mg/kg p.o.) and object recognition memory in mice (0.3–1 mg/kg p.o.). Rats trained to discriminate nicotine (0.4 mg/kg s.c.) from vehicle did not generalize to ABBF (0.3–30 mg/kg p.o.), suggesting that the nicotine cue is not mediated by the α7 nAChR and that selective α7 nAChR agonists may not share the abuse liability of nicotine. Our results support the hypothesis that α7 nAChR agonists may provide a novel therapeutic strategy for the treatment of cognitive deficits with low abuse potential.

Melittin and a chemically modified trichotoxin form alamethicin-type multi-state pores

The bee venom constituent, melittin, is structurally and functionally related to alamethicin. By forming solvent-free planar bilayers of small area (approx. 100 microns 2) on the tip of fire-polished glass pipettes we could observe single melittin pores in these membranes. An increase in the applied voltage induced further non-integral conductance levels. This indicates that melittin forms multi-level pores similar to those formed by alamethicin. Trichotoxin A40, an antibiotic analogue of alamethicin, also induces a voltage-dependent bilayer conductivity, but no stable pore states are resolved. However, chemical modification of the C-terminal molecule part by introduction of a dansyl group leads to a steeper voltage-dependence and pore state stabilization. Comparing structure and activity of several natural and synthetic amphiphilic polypeptides, we conclude that a lipophilic, N-terminal alpha-helical part of adequate length (dipole moment) and a large enough hydrophilic, C-terminal region are sufficient prerequisites for voltage-dependent formation of multi-state pores.

Ion channel reconstitution into lipid bilayer membranes on glass patch pipettes

Abstract Planar lipid bilayer membranes were formed at the tip of glass patch clamp pipettes, and ion channels reconstituted by fusion of protein-containing vesicles with the bilayer. The advantages of the method over conventional bilayer techniques are numerous and include better current resolution, improved bilayer stability, compatibility with patch clamp apparatus and simple handling. Completely solvent-free lipid bilayers without any pretreatment can be prepared and only small amounts of materials are required.

The Roboocyte: Automated cDNA/mRNA Injection and Subsequent TEVC Recording on Xenopus Oocytes in 96-Well Microtiter Plates

Membrane-bound neurotransmitter receptors and ion channels are among the most numerous and important drug targets, and electrophysiological methods are the gold standard for the study of their functional properties and their response to drugs. However, electrophysiological measurements are usually performed one at a time by highly skilled individuals, and secondary functional screening is often hampered by this lack of throughput. Accordingly, the use of automated procedures to increase the efficiency of electrophysiological techniques is of great interest. Among the many different electrophysiological techniques that have been described, two electrode voltage clamp recording (TEVC) from Xenopus oocytes seems particularly suitable for the implementation of automated measurement systems. Here, we describe a workstation that was expressly developed for this purpose. The Roboocyte is the first (and the only currently available) instrument that automatically performs both cDNA (or mRNA) injection and subsequent TEVC recording on Xenopus oocytes plated in a standard 96-well microtiter plate. This paper describes the scientific background of the oocyte expression system for drug screening and the development of the Roboocyte. Then, some technical details of the Roboocyte system are presented and, finally, results obtained with the Roboocyte are discussed with regard to increased throughput compared with manually performed experiments. Further information can be obtained at www.roboocyte.com.

A Novel Chloride Channel in Drosophila melanogaster Is Inhibited by Protons

A systematic analysis of the Drosophila genome data reveals the existence of pHCl, a novel member of ligand-gated ion channel subunits. pHCl shows nearly identical similarity to glutamate-, glycine-, and histamine-gated ion channels, does however not belong to any of these ion channel types. We identified three different sites, where splicing generates multiple transcripts of the pHCl mRNA. The pHCl is expressed in Drosophila embryo, larvae, pupae, and the adult fly. In embryos, in situ hybridization detected pHCl in the neural cord and the hindgut. Functional expression of the three different splice variants of pHCl in oocytes of Xenopus laevis and Sf9 cells induces a chloride current with a linear current-voltage relationship that is inhibited by extracellular protons and activated by avermectins in a pH-dependent manner. Further, currents through pHCl channels were induced by a raise in temperature. Our data give genetic and electrophysiological evidence that pHCl is a member of a new branch of ligand-gated ion channels in invertebrates with, however, a hitherto unique combination of pharmacological and biophysical properties.

Marine natural products acting on the acetylcholine-binding protein and nicotinic receptors: from computer modeling to binding studies and electrophysiology.

For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([125I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (Ki 0.5–1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH4C1 cell line, no competition with [125I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (Ki ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources.

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Background: Connexin 43 hemichannels participate in many cellular processes. To elucidate their location and function within living cells, they were labeled with GFP. Results: Recombinantly expressed Cx43 and Cx43-GFP form conducting hemichannels in reconstituted planar membranes. Their conductance states and voltage dependence differ. Conclusion: Fusion of GFP to Cx43 significantly affects the electrophysiological behavior of Cx43 hemichannels. Significance: GFP can significantly alter channel activities. In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants

Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117–119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs.

Single Hemichannels Recorded in Lipid Bilayers and Artificial Gap Junction Formation with Cells

Connexins (Cx) are members of a multigene family of membrane-spanning proteins that form gap junctions, which are composed of two hexameric hemichannels, called connexons. These gap junctions, organized in so-called gap junctional plaques, span the extracellular space/matrix of adjacent cells and thus allow a passive exchange of small molecules up to about 1 kDa. Connexins are widely distributed with various subtypes of connexin and are involved in different biological processes such transmission of information and propagation of action potential for e.g. Recent studies indicates that hemichannels do open under physiological and pathological conditions.In our study, we investigated the biophysical properties of hemichannels Cx26 and Cx43 which were isolated biochemically and reconstituted into synthetic lipid membranes. Both hemichannels are present in different tissues and involved in different pathologies. The results on a study of the Cx26 are presented. Reconstitutions of functional Cx26 and mutant hemichannels were performed. Secondly, Cx43 was purified and reconstituted into bilayers. The hemichannel Cx43 properties were compared to previous studies and showed similarities of conductance on single channel recordings of Cx43 in cells. Our focus was then to form artificial gap junctions, first between two unrelated cells and then between cells and bilayers containing functional hemichannels. This was done using Cx26 or Cx43. The bilayer-cell configuration allows to measure electrophysiological properties of the cells indirectly via gap junctions. Single channel recordings of gap junctions were recorded using a bilayer containing Cx43 and Cardiomyocytes expressing Cx43. Macroscopic currents were as well recorded between bilayers and cell lines expressing Cx26 or Cx43.