Bruce N. Cohen

Two mutations linked to nocturnal frontal lobe epilepsy cause use-dependent potentiation of the nicotinic ACh response

1 We constructed rat homologues (S252F and +L264) of two human α4 nicotinic mutations ‐ α4(S248F) and α4(777ins3) ‐ that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co‐expressed them with wild‐type rat β2 subunits in Xenopus oocytes. 2 The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use‐dependent potentiation of the response during a train of brief 100 nm ACh pulses. Second, they delayed the rise times of the 5–15 nm (+L264) and 30 nm (S252F) ACh responses. Third, they reduced extracellular Ca2+‐induced increases in the 30 μm ACh response. 3 Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage‐jump relaxations, enhanced steady‐state desensitization and reduced the single‐channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single‐channel conductance. Neither mutation affected the number of surface receptors measured by antibody binding but the S252F mutation reduced the maximum ACh response. 4 The ACh concentration dependence of use‐dependent potentiation and the delay in the rising phase of the mutant ACh response suggest that these effects are caused by a slow unblocking of the closed mutant receptors. Use‐dependent potentiation of the mutant response during a series of high‐frequency cholinergic inputs to the presynaptic terminal could trigger ADNFLE seizures by suddenly increasing nicotinic‐mediated transmitter release.

Pharmacological similarities between native brain and heterologously expressed α4β2 nicotinic receptors

We studied the pharmacological properties of native rat brain and heterologously expressed rat α4β2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti‐α4 antibody mAb 299. Immunodepletion with the anti‐β2 antibody mAb 270 showed that 89% of the mAb‐299‐precipitated rat brain receptors contained β2. The association and dissociation rate constants for 30 pM ±[3H]‐epibatidine binding to α4β2 receptors expressed in oocytes were 0.02±0.01 and 0.03±0.01 min−1 (±standard error, degrees of freedom=7–8) at 20–23°C. The Hill coefficients for ±[3H]epibatidine binding to the native brain, α4β2 receptors expressed in oocytes, and α4β2 receptors expressed in CV‐1 cells (using recombinant adenovirus) were 0.69–0.70 suggesting a heterogeneous receptor population. Fits of the ±[3H]‐epibatidine concentration‐binding data to a two‐site model gave KD s of 8–30 and 560–1,200 pM. The high‐affinity sites comprised 73–74% of the native brain and oocyte α4β2 receptor population, 85% of the CV‐1 α4β2 receptor population. The expression of rat α4β2 receptors in CV‐1 cells using vaccinia viral infection‐transfection resulted in a more homogeneous receptor population (Hill coefficient of 1.0±0.2). Fits of the ±[3H]‐epibatidine binding data to a single‐site model gave a KD of 40±3 pM. DHβE (IC50=260–470 nM) and the novel nicotine analogue NDNI (IC50=7–10 μM) inhibited 30 pM±[3H]‐epibatidine binding to the native brain and heterologously expressed α4β2 receptors equally well. The results show that α4β2‐containing nicotinic receptors in the rat brain and heterologously expressed rat α4β2 receptors have similar affinities for ±[3H]‐epibatidine, DHβE, and NDNI.

The α4 subunit of rat α4β2 nicotinic receptors is phosphorylated in vivo

Abstract The intracellular domains of the α4 and β2 neuronal nicotinic subunits between transmembrane segments 3 and 4 contain a number of predicted phosphorylation sites but there is no direct evidence that any of these sites are actually phosphorylated in vivo. We expressed rat α4β2 nicotinic receptors in Xenopus oocytes, labeled them by an overnight incubation in [ 32 P ]orthophosphate, and analyzed the immunoprecipitated receptors by autoradiography and Western blotting. Our results show that the oocytes contained three kinds of α4 subunits with apparent weights of 69, 79, and 89 kDa. The 89 kDa α4 subunit was the most heavily phosphorylated.

Functional characterization of a glutamate/aspartate transporter from the mosquito Aedes aegypti

SUMMARY Glutamate elicits a variety of effects in insects, including inhibitory and excitatory signals at both neuromuscular junctions and brain. Insect glutamatergic neurotransmission has been studied in great depth especially from the standpoint of the receptor-mediated effects, but the molecular mechanisms involved in the termination of the numerous glutamatergic signals have only recently begun to receive attention. In vertebrates, glutamatergic signals are terminated by Na+/K+-dependent high-affinity excitatory amino acid transporters (EAAT), which have been cloned and characterized extensively. Cloning and characterization of a few insect homologues have followed, but functional information for these homologues is still limited. Here we report a study conducted on a cloned mosquito EAAT homologue isolated from the vector of the dengue virus, Aedes aegypti. The deduced amino acid sequence of the protein, AeaEAAT, exhibits 40–50% identity with mammalian EAATs, and 45–50% identity to other insect EAATs characterized thus far. It transports l-glutamate as well as l- and d-aspartate with high affinity in the micromolar range, and demonstrates a substrate-elicited anion conductance when heterologously expressed in Xenopus laevis oocytes, as found with mammalian homologues. Analysis of the spatial distribution of the protein demonstrates high expression levels in the adult thorax, which is mostly observed in the thoracic ganglia. Together, the work presented here provides a thorough examination of the role played by glutamate transport in Ae. aegypti.

Dopamine D2-receptor activation elicits akinesia, rigidity, catalepsy, and tremor in mice expressing hypersensitive α4 nicotinic receptors via a cholinergic-dependent mechanism

Recent studies suggest that high‐affinity neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and β2 subunits (α4β2*) functionally interact with G‐protein‐coupled dopamine (DA) D2 receptors in basal ganglia. We hypothesized that if a functional interaction between these receptors exists, then mice expressing an M2 point mutation (Leu9′Ala) rendering α4 nAChRs hypersensitive to ACh may exhibit altered sensitivity to a D2‐receptor agonist. When challenged with the D2R agonist, quinpirole (0.5–10 mg/kg), Leu9′Ala mice, but not wild‐type (WT) litter‐mates, developed severe, reversible motor impairment characterized by rigidity, catalepsy, akinesia, and tremor. While striatal DA tissue content, baseline release, and quinpirole‐induced DA depletion did not differ between Leu9′Ala and WT mice, quinpirole dramatically increased activity of cholinergic striatal interneurons only in mutant animals, as measured by increased c‐Fos expression in choline acetyltransferase (ChAT)‐positive interneurons. Highlighting the importance of the cho‐linergic system in this mouse model, inhibiting the effects of ACh by blocking muscarinic receptors, or by selectively activating hypersensitive nAChRs with nicotine, rescued motor symptoms. This novel mouse model mimics the imbalance between striatal DA/ACh function associated with severe motor impairment in disorders such as Parkinsons disease, and the data suggest that a D2R‐α4*‐nAChR functional interaction regulates cholinergic interneuron activity.—Zhao‐Shea, R., Cohen, B. N., Just, H., McClure‐Begley, T., Whiteaker, P., Grady, S. R., Salminen, O., Gardner, P. D., Lester, H. A., Tapper, A. R. Dopamine D2‐receptor activation elicits akinesia, rigidity, catalepsy, and tremor in mice expressing hypersensitive α4 nicotinic receptors via a cholinergic‐dependent mechanism. FASEB J. 24, 49–57 (2010). www.fasebj.org

The β subunit dominates the relaxation kinetics of heteromeric neuronal nicotinic receptors

1 The ACh‐induced voltage‐jump relaxation currents of the nicotinic receptors formed by pair‐wise expression of the rat α2, α3, or α4 subunits with the β2 or β4 subunit in Xenopus oocytes were fitted best by the sum of two exponentials and a constant between ‐60 and ‐150 mV. 2 As the ACh concentration approached zero, the relaxation time constants approached limiting values that should equal the single‐channel burst duration at low ACh concentrations and the synaptic current decay time constants. β4 co‐expression prolonged the zero ACh concentration limits for the relaxation time constants. The fast β4 zero ACh concentration limits ranged from 40 to 121 ms between ‐60 and ‐150 mV, and the slow β4 zero ACh concentration limits ranged from 274 to 1039 ms. In contrast, the fast β2 limits were 4–6 ms over the same voltage range and the slow β2 limits were 30–53 ms. 3 Expression with the β4 subunit increased the voltage sensitivity of the α2, α3 and slow α4 relaxation time constants but not that of the fast α4 relaxation time constant. 4 Reducing the temperature from 22°C to 8–9°C increased the α4β2 and α3β4 relaxation time constants 2.3‐ to 6.6‐fold and reduced the fractional amplitude of the fast relaxation component. It also increased the voltage dependence of the fast α3β4 relaxation time constant and decreased that of the slow time constant. The Q10 for α4β2 and α3β4 relaxation time constants ranged from 1.9 to 3.9 between 10 and 20°C. 5 The β subunit appears to have a dominant influence on the voltage‐jump relaxation kinetics of heteromeric neuronal nicotinic receptors.

Mutation Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Reduces Low-Sensitivity α4β2, and Increases α5α4β2, Nicotinic Receptor Surface Expression

A number of mutations in α4β2-containing (α4β2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the β2 subunit called β2V287L. Two α4β2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (α4)2(β2)3 subunit stoichiometry and a low-sensitivity subtype with (α4)3(β2)2 stoichiometry. The α5 nicotinic subunit also co-assembles with α4β2 to form a high-sensitivity α5α4β2 nAChR. Previous studies suggest that the β2V287L mutation suppresses low-sensitivity α4β2* nAChR expression in a knock-in mouse model and also that α5 co-expression improves the surface expression of ADNFLE mutant nAChRs in a cell line. To test these hypotheses further, we expressed mutant and wild-type (WT) nAChRs in oocytes and mammalian cell lines, and measured the effects of the β2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The β2V287L mutation reduced the EC50 values of high- and low-sensitivity α4β2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity α4β2 expression. In contrast, it did not affect the EC50 of α5α4β2 nAChRs for ACh. Measurements of the ACh responses of WT and mutant nAChRs expressed in mammalian cell lines using a voltage-sensitive fluorescent dye and whole-cell patch-clamping confirm the oocyte data. They also show that, despite reducing the maximum response, β2V287L increased the α4β2 response to a sub-saturating ACh concentration (1 μM). Finally, imaging SEP-tagged α5, α4, β2, and β2V287L subunits showed that β2V287L reduced total α4β2 nAChR surface expression, increased the number of β2 subunits per α4β2 receptor, and increased surface α5α4β2 nAChR expression. Thus, the β2V287L mutation alters the subunit composition and sensitivity of α4β2 nAChRs, and increases α5α4β2 surface expression.

Pharmacological differences between immunoisolated native brain and heterologously expressed rat α4β2 nicotinic receptors

Abstract Native brain and heterologously expressed rat α4β2 nicotinic receptors (in Xenopus oocytes and CV-1 cells) were immunoisolated with the anti-α4 antibody mAb 299 and their pharmacological properties were compared using [ 3 H](±)epibatidine, the novel N-alkylnicotinium analog N - n -octylnicotinium iodide (NONI), and the ganglionic antagonist trimethaphan (TRM). The equilibrium dissociation constant ( K d ) for [ 3 H](±)epibatidine binding to the native and heterologously expressed receptors ranged from 13 to 21 pM. The Hill coefficients for [ 3 H](±)epibatidine binding to the native and expressed receptors ranged from 0.8 to 1.1 and were consistent with a single high-affinity site. NONI inhibited 30 pM [ 3 H](±)epibatidine binding to the native and expressed receptors with similar potency (IC 50 values of 6–7 μM). However, [ 3 H](±)epibatidine dissociated 2–3 times more slowly from the native, than from the expressed receptors and TRM inhibited 30 pM [ 3 H](±)epibatidine binding to the native receptors (IC 50 value of 330 μM) less potently than it did to the receptors expressed in oocytes (IC 50 value of 16 μM) or CV-1 cells (IC 50 value of 55 μM). The differences between the native and expressed [ 3 H](±)epibatidine dissociation rate constants and IC 50 values for TRM were significant for both host cell types, although the values for the CV-1-expressed receptors were closer to the native ones than were those for the oocyte-expressed receptors. Thus, the epibatidine and trimethaphan binding sites in native and expressed α4β2 receptors appear to have significantly different structural or chemical properties.

A recombinant adenovirus that directs secretion of biologically active κ-bungarotoxin from mammalian cells

A novel Cre-lox system was used to construct an adenovirus encoding kappa-bungarotoxin (kappa-Bgt), modified to be secreted by attachment of a bovine prolactin signal sequence at the N-terminus of the toxin. Western blot of medium from HEK-293 cells infected with the virus demonstrated that recombinant kappa-Bgt (R-kappa-Bgt) was secreted. The biological activity of the secreted R-kappa-Bgt was investigated in Xenopus oocytes that expressed neuronal nicotinic acetylcholine receptor (nAChR) subtypes alpha3beta2 and alpha2beta2. The recombinant toxin inhibited the response of alpha3beta2 type AChRs to ACh, but did not inhibit the response of alpha2beta2 type AChRs. These data demonstrated that the recombinant adenovirus directs the secretion of biologically active kappa-Bgt from a mammalian cell line. Because adenovirus can be used to infect post-mitotic cells, recombinant adenoviruses encoding biologically active peptides may be of use as delivery vehicles for in vivo experiments where repeated application of the purified peptide is unfeasible.