Gregg B. Wells

Assembly of Human Neuronal Nicotinic Receptor α5 Subunits with α3, β2, and β4 Subunits

Nicotinic acetylcholine receptors formed from combinations of α3, β2, β4, and α5 subunits are found in chicken ciliary ganglion neurons and some human neuroblastoma cell lines. We studied the co-expression of various combinations of cloned human α3, β2, β4, and α5 subunits in Xenopus oocytes. Expression on the surface membrane was found only for combinations of α3β2, α3β4, α3β2α5, and α3β4α5 subunits but not for other combinations of one, two, or three of these subunits. α5 subunits assembled inside the oocyte with β2 but not with α3 subunits or other α5 subunits. α5 subunits coassembled very efficiently with α3β2 or α3β4 combinations. The presence of α5 subunits had very little effect on the binding affinities for epibatidine of receptors containing also α3 and β2 or α3 and β4 subunits. The presence of α5 subunits increased the rate of desensitization of both receptors containing also α3 and β2 or α3 and β4 subunits. In the case of receptors containing α3 and β4 subunits, the addition of α5 subunits had little effect on the responses to acetylcholine or nicotine. However, in the case of receptors containing α3 and β2 subunits, the addition of α5 subunits reduced the EC50 for acetylcholine from 28 to 0.5 μM and the EC50 for nicotine from 6.8 to 1.9 μM, while increasing the efficacy of nicotine from 50% on α3β2 receptors to 100% on α3β2α5 receptors. Both α3β2 and α3β2α5 receptors expressed in oocytes sedimented at the same 11 S value as native α3-containing receptors from the human neuroblastoma cell line SH-SY5Y. In the receptors from the neuroblastoma α3, β2, and α5 subunits were co-assembled, and 56% of the receptor subtypes containing α3 subunits also contained β2 subunits. The β2 subunit-containing receptors from SH-SY5Y cells exhibited the high affinity for epibatidine characteristic of receptors formed from α3 and β2 or α3, β2, and α5 subunits rather than the low affinity exhibited by receptors formed from α3 and β4 or α3, β4, and α5 subunits. Nicotine, like the structurally similar toxin epibatidine, also distinguishes by binding affinity two subtypes of receptors containing α3 subunits in SH-SY5Y cells. The affinities of α3β2 receptors expressed in oocytes were similar to the affinities of native α3 containing receptors from SH-SY5Y cells for acetylcholine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium.

Chapter 10 Structure and function of neuronal nicotinic acetylcholine receptors

Publisher Summary This chapter discusses some aspects of neuronal acetylcholine receptors (AChRs), reviews some of the recent studies of the mechanism by which chronic exposure to nicotine affects aα4β2 AChRs, and describes some of the pharmacological properties of neuronal AChRs, especially the differences between α7 and α8 AChRs and the usefulness of epibatidine (exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.l]-heptane) as a ligand for many types of neuronal AChRs. The basic homologies in structure of receptors in this superfamily have been illustrated in the chapter. Homologies in overall domain relationships are demonstrated by showing that functional mosaics could be made in which the large N-terminal extracellular domain of α7 AChRs could be grafted just before the first transmembrane domain to the C-terminal part of 5HT 3 receptors. This resulted in receptors with acetylcholine-gated cation channels having the ion selectivity of 5HT 3 receptors. Close similarities in the overall structures of the ion channels are demonstrated by showing that changing only three amino acids in the sequence lining the cation-specific channel of excitatory α7 neuronal AChRs to amino acids typical of the anion-specific channels of inhibitory glycine or GABA A receptors changed the ion selectivity of the mutated a7 AChR channels from cations to anions

Genomic basis for the convergent evolution of electric organs

Only one way to make an electric organ? Electric fish have independently evolved electric organs that help them to communicate, navigate, hunt, and defend themselves. Gallant et al. analyzed the genome of the electric eel and the genes expressed in two other distantly related electric fish. The same genes were recruited within the different species to make evolutionarily new structures that function similarly. Science, this issue p. 1522 Multiple divergent fish lineages have used the same evolutionary toolkit to produce electric organs. Little is known about the genetic basis of convergent traits that originate repeatedly over broad taxonomic scales. The myogenic electric organ has evolved six times in fishes to produce electric fields used in communication, navigation, predation, or defense. We have examined the genomic basis of the convergent anatomical and physiological origins of these organs by assembling the genome of the electric eel (Electrophorus electricus) and sequencing electric organ and skeletal muscle transcriptomes from three lineages that have independently evolved electric organs. Our results indicate that, despite millions of years of evolution and large differences in the morphology of electric organ cells, independent lineages have leveraged similar transcription factors and developmental and cellular pathways in the evolution of electric organs.

Steady-State Adaptation of Mechanotransduction Modulates the Resting Potential of Auditory Hair Cells, Providing an Assay for Endolymph [Ca2+]

The auditory hair cell resting potential is critical for proper translation of acoustic signals to the CNS, because it determines their filtering properties, their ability to respond to stimuli of both polarities, and, because the hair cell drives afferent firing rates, the resting potential dictates spontaneous transmitter release. In turtle auditory hair cells, the filtering properties are established by the interactions between BK calcium-activated potassium channels and an L-type calcium channel (electrical resonance). However, both theoretical and in vitro recordings indicate that a third conductance is required to set the resting potential to a point on the ICa and IBK activation curves in which filtering is optimized like that found in vivo. Present data elucidate a novel mechanism, likely universal among hair cells, in which mechanoelectric transduction (MET) and its calcium-dependent adaptation provide the depolarizing current to establish the hair cell resting potential. First, mechanical block of the MET current hyperpolarized the membrane potential, resulting in broadband asymmetrical resonance. Second, altering steady-state adaptation by altering the [Ca2+] bathing the hair bundle changed the MET current at rest, the magnitude of which resulted in membrane potential changes that encompassed the best resonant voltage. The Ca2+ sensitivity of adaptation allowed for the first physiological estimate of endolymphatic Ca2+ near the MET channel (56 ± 11 μm), a value similar to bulk endolymph levels. These effects of MET current on resting potential were independently confirmed using a theoretical model of electrical resonance that included the steady-state MET conductance.

Water-soluble nicotinic acetylcholine receptor formed by alpha7 subunit extracellular domains.

Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the α7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression inXenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for α-bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the α7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length α7 AChR.

Suprasellar osteolipoma : Case report

BACKGROUND Osteolipomas are distinguished from other intracranial lipomas by their arrangement of central adipose and peripheral osseous tissues and by characteristically arising in the suprasellar/interpeduncular region. METHODS We report computed tomography (CT), magnetic resonance imaging (MRI), and pathology findings from this 34-year-old man who underwent surgical removal of this benign lesion. RESULTS This case displays the distinctive histopathology that has been reported in 13 of 31 (42%) lipomas in this region. In contrast, ossification of lipomas at other intracranial sites is relatively rare. CONCLUSIONS Ossification should be expected in many suprasellar/interpeduncular lipomas, and osteolipoma should be included in the radiologic differential diagnosis of fat-intensity masses with calcification in this region.

Structural answers and persistent questions about how nicotinic receptors work.

The electron diffraction structure of nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata and the X-ray crystallographic structure of acetylcholine binding protein (AChBP) are providing new answers to persistent questions about how nAChRs function as biophysical machines and as participants in cellular and systems physiology. New high-resolution information about nAChR structures might come from advances in crystallography and NMR, from extracellular domain nAChRs as high fidelity models, and from prokaryotic nicotinoid proteins. At the level of biophysics, structures of different nAChRs with different pharmacological profiles and kinetics will help describe how agonists and antagonists bind to orthosteric binding sites, how allosteric modulators affect function by binding outside these sites, how nAChRs control ion flow, and how large cytoplasmic domains affect function. At the level of cellular and systems physiology, structures of nAChRs will help characterize interactions with other cellular components, including lipids and trafficking and signaling proteins, and contribute to understanding the roles of nAChRs in addiction, neurodegeneration, and mental illness. Understanding nAChRs at an atomic level will be important for designing interventions for these pathologies.

Ion selectivity predictions from a two-site permeation model for the cyclic nucleotide-gated channel of retinal rod cells

We developed a two-site, Eyring rate theory model of ionic permeation for cyclic nucleotide-gated channels (CNGCs). The parameters of the model were optimized by simultaneously fitting current-voltage (IV) data sets from excised photoreceptor patches in electrolyte solutions containing one or more of the following ions: Na+, Ca2+, Mg2+, and K+. The model accounted well for 1) the shape of the IV relations; 2) the binding affinity for Na+; 3) reversal potential values with single-sided additions of Ca2+ or Mg2+ and biionic KCl; and 4) the K1 and voltage dependence for divalent block from the cytoplasmic side of the channel. The differences between the predicted K1s for extracellular block by Ca2+ and Mg2+ and the values obtained from heterologous expression of only the alpha-subunit of the channel suggest that the beta-subunit or a cell-specific factor affects the interaction of divalent cations at the external but not the internal face of the channel. The model predicts concentration-dependent permeability ratios with single-sided addition of Ca2+ and Mg2+ and anomalous mole fraction effects under a limited set of conditions for both monovalent and divalent cations. Ca2+ and Mg2+ are predicted to carry 21% and 10%, respectively, of the total current in the retinal rod cell at -60 mV.

A Highly Conserved Cytoplasmic Cysteine Residue in the α4 Nicotinic Acetylcholine Receptor Is Palmitoylated and Regulates Protein Expression

Background: The mechanisms underlying nicotinic acetylcholine receptor (nAChR) trafficking are unclear. Results: Cysteine mutations within cytoplasmic loops of the α4 nAChR subunit change surface and total receptor expression, and a cysteine in the first loop is palmitoylated. Conclusion: α4 nAChR intracellular cysteines influence receptor stability and trafficking. Significance: Identifying the determinants of nAChR trafficking will provide insight into nAChR biology. Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [3H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1–M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [3H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4β2 nAChR.

The effects of ibogaine on dopamine and serotonin transport in rat brain synaptosomes.

Ibogaine has been shown to affect biogenic amine levels in selected brain regions. Because of the involvement of these neurotransmitters in drug addiction, the effects of ibogaine on biogenic amine transport may contribute to the potential anti-addictive properties of ibogaine in vivo. With rat brain synaptosomes as our experimental system, we measured the effects of ibogaine on the uptake and release of dopamine (DA) and serotonin (5-HT). Ibogaine competitively blocked both DA and 5-HT uptake with IC50 values of 20 microM at 75 nM 3H-DA and 2.6 microM at 10 nM 3H-5-HT. Ibogaine had no effect on K+-induced release of 3H-DA from preloaded synaptosomes, but 20 microM and 50 microM ibogaine inhibited roughly 40% and 60%, respectively, of the K(+)-induced release of 3H-5-HT from preloaded synaptosomes. In the absence of a depolarizing stimulus, ibogaine evoked a small release of 3H-DA but not 3H-5-HT. These relatively low-potency effects of ibogaine on DA and 5-HT uptake in synaptosomes are consistent with the low binding affinity of ibogaine that has been previously reported for DA and 5-HT transporters. Our results show that if ibogaine modulates DA and 5-HT levels in the brain by directly blocking their uptake, then a concentration of ibogaine in the micromolar range is required. Furthermore, if the anti-addictive effects of ibogaine require this concentration, then ibogaine likely exerts these effects through a combination of neurotransmitter pathways, because binding affinities and functional potencies of ibogaine in the micromolar range have been reported for a variety of neuronal receptors and transporters.