nAChR Currents Research Articles

Chrna5 genotype determines the long-lasting effects of developmental in vivo nicotine exposure on prefrontal attention circuitry

Maternal smoking during pregnancy repeatedly exposes the developing fetus to nicotine and is linked with attention deficits in offspring. Corticothalamic neurons within layer VI of the medial prefrontal cortex are potential targets in the disruption of attention circuitry by nicotine, a process termed teratogenesis. These prefrontal layer VI neurons would be likely targets because they are developmentally excited and morphologically sculpted by a population of nicotinic acetylcholine receptors (nAChRs) that are sensitive to activation and/or desensitization by nicotine. The maturational effects of these α4β2* nAChRs and their susceptibility to desensitization are both profoundly altered by the incorporation of an α5 subunit, encoded by the chrna5 gene. Here, we investigate nicotine teratogenesis in layer VI neurons of wildtype and α5−/− mice. In vivo chronic nicotine exposure during development significantly modified apical dendrite morphology and nAChR currents, compared with vehicle control. The direction of the changes was dependent on chrna5 genotype. Surprisingly, neurons from wildtype mice treated with in vivo nicotine resembled those from α5−/− mice treated with vehicle, maintaining into adulthood a morphological phenotype characteristic of immature mice together with reduced nAChR currents. In α5−/− mice, however, developmental in vivo nicotine tended to normalize both adult morphology and nAChR currents. These findings suggest that chrna5 genotype can determine the effect of developmental in vivo nicotine on the prefrontal cortex. In wildtype mice, the lasting alterations to the morphology and nAChR activation of prefrontal layer VI neurons are teratogenic changes consistent with the attention deficits observed following developmental nicotine exposure.

The α5 Subunit Regulates the Expression and Function of α4*-Containing Neuronal Nicotinic Acetylcholine Receptors in the Ventral-Tegmental Area

Human genetic association studies have shown gene variants in the α5 subunit of the neuronal nicotinic receptor (nAChR) influence both ethanol and nicotine dependence. The α5 subunit is an accessory subunit that facilitates α4* nAChRs assembly in vitro. However, it is unknown whether this occurs in the brain, as there are few research tools to adequately address this question. As the α4*-containing nAChRs are highly expressed in the ventral tegmental area (VTA) we assessed the molecular, functional and pharmacological roles of α5 in α4*-containing nAChRs in the VTA. We utilized transgenic mice α5+/+(α4YFP) and α5-/-(α4YFP) that allow the direct visualization and measurement of α4-YFP expression and the effect of the presence (α5+/+) and absence of α5 (-/-) subunit, as the antibodies for detecting the α4* subunits of the nAChR are not specific. We performed voltage clamp electrophysiological experiments to study baseline nicotinic currents in VTA dopaminergic neurons. We show that in the presence of the α5 subunit, the overall expression of α4 subunit is increased significantly by 60% in the VTA. Furthermore, the α5 subunit strengthens baseline nAChR currents, suggesting the increased expression of α4* nAChRs to be likely on the cell surface. While the presence of the α5 subunit blunts the desensitization of nAChRs following nicotine exposure, it does not alter the amount of ethanol potentiation of VTA dopaminergic neurons. Our data demonstrates a major regulatory role for the α5 subunit in both the maintenance of α4*-containing nAChRs expression and in modulating nicotinic currents in VTA dopaminergic neurons. Additionally, the α5α4* nAChR in VTA dopaminergic neurons regulates the effect of nicotine but not ethanol on currents. Together, the data suggest that the α5 subunit is critical for controlling the expression and functional role of a population of α4*-containing nAChRs in the VTA.

Whole-cell patch-clamp recording of nicotinic acetylcholine receptors in adult Brugia malayi muscle

Lymphatic filariasis is a debilitating disease caused by clade III parasites like Brugia malayi and Wuchereria bancrofti. Current recommended treatment regimen for this disease relies on albendazole, ivermectin and diethylcarbamazine, none of which targets the nicotinic acetylcholine receptors in these parasitic nematodes. Our aim therefore has been to develop adult B. malayi for electrophysiological recordings to aid in characterizing the ion channels in this parasite as anthelmintic target sites. In that regard, we recently demonstrated the amenability of adult B. malayi to patch-clamp recordings and presented results on the single-channel properties of nAChR in this nematode. We have built on this by recording whole-cell nAChR currents from adult B. malayi muscle. Acetylcholine, levamisole, pyrantel, bephenium and tribendimidine activated the receptors on B. malayi muscle, producing robust currents ranging from >200pA to ~1.5nA. Levamisole completely inhibited motility of the adult B. malayi within 10min and after 60min, motility had recovered back to control values.

1606 FUNCTIONAL PLASTICITY OF AUTONOMIC PELVIC GANGLIA IN RAT MODELS OF OVERACTIVE BLADDER

INTRODUCTION AND OBJECTIVES: Bladder outlet obstruction (BOO) in benign prostatic hyperplasia (BPH), a common problem affecting elderly men, caused urinary symptoms such as urgency, frequency, incomplete bladder emptying, and weak urine flow. Overactive bladder (OAB) is a syndrome characterized by urinary urgency with frequency and nocturia. Peripheral autonomic major pelvic ganglion (MPG) neurons are essential for the generation of storage and voiding reflexes. Under certain pathophysiological conditions such as spinal cord injury and chronic cystitis, plastic alterations in MPG neurons may cause abnormal functions of the bladder. But neurogenic mechanisms underlying OAB remain poorly understood. The hypothesis of the present study is that OAB is associated with changes in functions of MPG and dorsal roots ganglion (DRG) neurons in bladder outlet obstruction (BOO) rat models. METHODS: We were examined whether partial BOO alters expression and activity of nicotinic acetylcholine receptors (nAChRs) involved in synaptic transmission within MPG, and excitability of MPG and DRG neurons innervating the urinary bladder. Toward this end, rat models of partial urethral obstruction (PUO) and BPH were produced by partial urethra ligation and sc injection of testosterone/17-ƒo-estradiol, respectively. Cystometry were performed, indicating development of OAB. Real-time PCR analysis showed that the nAChRs ƒN3 and £4 subunits were up-regulated in MPG neurons of BOO rats. Then, nAChR currents were measured in DiI-labeled sympathetic and parasympathetic MPG neurons innervating the bladder detrusor muscles. RESULTS: nAChR current densities were significantly increased in parasympathetic MPG neurons, but not in sympathetic PG neurons. Under the current-clamp mode of the patch-clamp technique, action potentials were recorded in DRG and MPG neurons of control and BOO rats. BOO produced hyperexcitability of the bladder DRG and MPG neurons via reducing rheobase and AHP duration. One of the ionic mechanisms underlying the hyperexcitability is up-regulation of T-type Ca2 channels expressed in DRG and sympathetic MPG neurons. The decreased AHP duration also suggest that expression of Ca2 -activated Cland/or K (i.e., SK channels) might be altered in the bladder DRG and MPG neurons of BOO rats. CONCLUSIONS: BOO-induced OAB might be associated with enhanced ganglionic transmission and/or hyperexcitability of peripheral autonomic motor and sensory neurons innervating the bladder. Clinically, use of T-type Ca2 channel blockers might be a potential option of pharmacologic management of OAB.

Layer-Specific Interference with Cholinergic Signaling in the Prefrontal Cortex by Smoking Concentrations of Nicotine

Adolescence is a period in which the developing prefrontal cortex (PFC) is sensitive to maladaptive changes when exposed to nicotine. Nicotine affects PFC function and repeated exposure to nicotine during adolescence impairs attention performance and impulse control during adulthood. Nicotine concentrations experienced by smokers are known to desensitize nicotinic acetylcholine receptors (nAChRs), but the impact thereof on PFC circuits is poorly understood. Here, we investigated how smoking concentrations of nicotine (100-300 nm) interfere with cholinergic signaling in the mouse PFC. nAChR desensitization depends on subunit composition. Since nAChR subunits are differentially expressed across layers of the PFC neuronal network, we hypothesized that cholinergic signaling through nAChRs across layers would suffer differentially from exposure to nicotine. Throughout the PFC, nicotine strongly desensitized responses to ACh in neurons expressing β2* nAChRs, whereas ACh responses mediated by α7 nAChRs were not hampered. The amount of desensitization of β2* nAChR currents depended on neuron type and cortical layer. β2*-mediated responses of interneurons in LII-III and LVI completely desensitized, while cholinergic responses in LV interneurons and LVI pyramidal cells showed less desensitization. This discrepancy depended on α5 subunit expression. Two-photon imaging of neuronal population activity showed that prolonged exposure to nicotine limited cholinergic signaling through β2* nAChRs to deep PFC layers where α5 subunits were expressed. Together, our results demonstrate a layer-dependent decrease in cholinergic activation of the PFC through nAChRs by nicotine. These mechanisms may be one of the first steps leading up to the pathophysiological changes associated with nicotine exposure during adolescence.

Effect of Bispiridinium Compounds on Human Nicotinic α7 Receptor Currents

Nicotinic acetylcholine receptors (nAChRs) are important targets in drug discovery due to their high physiological importance in pathogenesis and poisoning. Life threatening poisoning by organophosphorus compounds results in over-stimulation of cholinergic receptors by blocking acetylcholinesterase (AChE). Antidotal therapy is based on reactivation of inhibited AChE by oximes, i.e. obidoxime. A direct interaction of bispyridinium oximes with nicotinic receptors was postulated in rodent studies, but could not be verified in human tissues. Therefore this study investigated interactions of bispyridinium oximes with neuronal human nicotinic α7 receptor.Human α7 nAChR were expressed in stably transfected rat pituitary tumor cells (GH4C1). Whole-cell patch clamping was performed with planar electrodes in an 8-channel Patchliner system (Nanion Technologies GmbH, Munich, Germany). Cholinergic currents were activated by nicotine and modulated by PNU 120596. The dose-response relationship of the bispyridinium oximes obidoxime and HI 6 was measured in concentrations between 0.01-100 μM.Nicotinic induced inward currents stopped within a few ms, because of receptor desensitisation. This desensitisation could be prevented by adding positive allosteric modulator PNU 120596. Under such conditions, HI 6 reduced the cation inward current of human nicotinic α7 receptors with an IC50 of 83 ± 30 nM. In contrast, nicotinic activation of the human α7 subtype was not affected by obidoxime in μM range. Without PNU 120596 both HI 6 and obidoxime enhanced nicotinic inward current of the human α7 receptor.These results demonstrated that HI 6 and obidoxime interact with the human nicotinic α7 receptor. As PNU 120596 the bispyridinium-oximes can not only increase the maximal amplitude and potency of nicotinic evoked α7 nAChR currents, but also suppress desensitization. This suggests an allosteric effect of these substances. The therapeutic consequences in cases of poisoning by organophosphorus compounds has yet to be tested.

Amyloid β‐protein suppressed nicotinic acetylcholine receptor‐mediated currents in acutely isolated rat hippocampal CA1 pyramidal neurons

Amyloid β protein (Aβ) is responsible for the deficits of learning and memory in Alzheimer's disease (AD). The high affinity between Aβ and nicotinic acetylcholine receptors (nAChRs) suggests that the impairment of cognitive function in AD might be involved in the Aβ-induced damage of nAChRs. This study investigated the effects of Aβ fragments on nAChR-mediated membrane currents in acutely isolated rat hippocampal pyramidal neurons by using whole-cell patch clamp technique. The results showed that: (1) nonspecific nAChR agonist nicotine, selective α7 nAChR agonist choline, and α4β2 nAChR agonist epibatidine all effectively evoked inward currents in CA1 neurons at normal resting membrane potential, with different desensitization characteristics; (2) acute application of different concentrations (pM-μM) of Aβ25-35, Aβ31-35, or Aβ35-31 alone did not trigger any membrane current, but pretreatment with 1 μM Aβ25-35 and Aβ31-35 similarly and reversibly suppressed the nicotine-induced currents; (3) further, choline- and epibatidine-induced currents were also reversibly suppressed by the Aβ pretreatment, but more prominent for the choline-induced response. These results demonstrate that the functional activity of both α7 and α4β2 nAChRs in the membrane of acutely isolated hippocampal neurons was significantly downregulated by Aβ treatment, suggesting that nAChRs, especially α7 nAChRs, in the brain may be the important biological targets of neurotoxic Aβ in AD. In addition, the similar suppression of nAChR currents by Aβ25-35 and Aβ31-35 suggests that the sequence 31-35 in Aβ molecule may be a shorter active center responsible for the neurotoxicity of Aβ in AD.

Lack of Modulation of Nicotinic Acetylcholine Alpha-7 Receptor Currents by Kynurenic Acid in Adult Hippocampal Interneurons

Kynurenic acid (KYNA), a classical ionotropic glutamate receptor antagonist is also purported to block the α7-subtype nicotinic acetylcholine receptor (α7* nAChR). Although many published studies cite this potential effect, few have studied it directly. In this study, the α7*-selective agonist, choline, was pressure-applied to interneurons in hippocampal subregions, CA1 stratum radiatum and hilus of acute brain hippocampal slices from adolescent to adult mice and adolescent rats. Stable α7* mediated whole-cell currents were measured using voltage-clamp at physiological temperatures. The effects of bath applied KYNA on spontaneous glutamatergic excitatory postsynaptic potentials (sEPSC) as well as choline-evoked α7* currents were determined. In mouse hilar interneurons, KYNA totally blocked sEPSC whole-cell currents in a rapid and reversible manner, but had no effect on choline-evoked α7* whole-cell currents. To determine if this lack of KYNA effect on α7* function was due to regional and/or species differences in α7* nAChRs, the effects of KYNA on choline-evoked α7* whole-cell currents in mouse and rat stratum radiatum interneurons were tested. KYNA had no effect on either mouse or rat stratum radiatum interneuron choline-evoked α7* whole-cell currents. Finally, to test whether the lack of effect of KYNA was due to unlikely slow kinetics of KYNA interactions with α7* nAChRs, recordings of a7*-mediated currents were made from slices that were prepared and stored in the presence of 1 mM KYNA (>90 minutes exposure). Under these conditions, KYNA had no measurable effect on α7* nAChR function. The results show that despite KYNA-mediated blockade of glutamatergic sEPSCs, two types of hippocampal interneurons that express choline-evoked α7* nAChR currents fail to show any degree of modulation by KYNA. Our results indicate that under our experimental conditions, which produced complete KYNA-mediated blockade of sEPSCs, claims of KYNA effects on choline-evoked α7* nAChR function should be made with caution.

Pharmacological characterization of native α7 nicotinic ACh receptors and their contribution to depolarization‐elicited exocytosis in human chromaffin cells

Expression of α7 nicotinic acetylcholine receptors (nAChRs) and their role in exocytosis have not yet been examined in human chromaffin cells. To characterize these receptors and investigate their function, patch-clamp experiments were performed in human chromaffin cells from organ donors. The nicotinic current provoked by 300µM ACh in voltage-clamped cells was blocked by the nicotinic receptor antagonists α-bungarotoxin (α-Bgtx; 1µM; 6 ± 1.7%) or methyllycaconitine (MLA; 10nM; 7 ± 1.6%), respectively, in an irreversible and reversible manner, without affecting exocytosis. Choline (10mM) pulses induced a biphasic current with an initial quickly activated (5.5 ± 0.4ms rise time) and inactivated component (8.5 ± 0.4ms time constant) (termed α7), which was blocked by α-Bgtx or MLA, followed by a slower component (non-α7). α7 nAChR currents were dissected by blocking the non-α7 nAChR current component of the ACh and choline response with the α6* nAChR blocker α-conotoxin (α-Ctx) MII[S4A, E11A, L15A]. PNU-282987, an α7 nAChR-specific agonist, elicited rapidly activated and rapidly inactivated currents. α7 nAChR-positive allosteric modulators, such as 5-hydroxyindole (1mM) and PNU-120596 (10µM), potentiated responses that were blocked by α-Bgtx or MLA. Exocytosis was evoked by depolarization-elicited α7 nAChR currents, using choline in the presence of α-Ctx MII[MS4A, E11A, L15A] or PNU-282987 as agonists. Our electrophysiological recordings of pure α7 nAChR currents elicited by rapid application of agonists demonstrated that functional α7 nAChRs are expressed and contribute to depolarization-elicited exocytosis in human chromaffin cells.

Genistein’s Effects on Alpha7 nAChR Currents Depends upon Exposure Duration and Time Course of Expression in Xenopus Oocytes

Several previous studies that explored tyrosine phosphorylation/dephosphorylation-involvement in functional upregulation of α7-nAChRs found that genistein, a broad-spectrum inhibitor of protein tyrosine kinases (PTKs), increased Ach-evoked α7-nAChR currents. Interpretation of genistein's effects as due to perturbation of tyrosine-phosphorylation processes is complicated, however, by subsequent findings that genistein is a strong positive allosteric modulator (PAM) of α7-nAChRs. Our study asked if genistein's enhancement of α7-nAChRs currents is entirely attributable to PAM activity, or if non-PAM effects contribute as well. We compared the effects of brief (1 min) vs. extended (24 h) exposure of oocytes expressing α7-nAChRs to genistein (100 μM), both 2 and 5 days following cRNA injections (2.0 ng). On day 2, the Ach-evoked (500 μM) peak currents for control cells, and cells exposed for 1 min, or 24 h, to genistein were (mean ± SEM, nA): 29.32 ± 12.10 (n=24), 130.95 ± 35.42 (n=22), and 374 ± 105.21 (n=8). On day 5, the comparable values were: 108.61 ± 14.85 (n=10), 310.81 ± 56.22 (n=9), and 605.00 ± 128.03 nA (n=11). On day 2, 1 min genistein caused a 4.5 fold-facilitation; 24 h exposure caused a 12.8 fold-facilitation. On day 5, 1 min genistein caused a 2.4 fold-facilitation; 24 h genistein caused a 5.6 fold-facilitation. Thus, genistein-enhancement of α7 currents varied inversely with the degree of functional α7-nAChR expression, being greater for day 2 than 5. Genistein's effect on desensitization of α7-nAChR currents was also separable from facilitation. On day 2, genistein increased peak current without affecting desensitization. On day 5, genistein increased peak current and slowed desensitization. Our results suggest that genistein's enhancement of α7-nAChR currents is not entirely due to PAM effects, but involves other mechanisms too, particularly on day 2.

α7-Containing and Non-α7-Containing Nicotinic Receptors Respond Differently to Spillover of Acetylcholine

We explored whether nicotinic acetylcholine receptors (nAChRs) might participate in paracrine transmission by asking if they respond to spillover of ACh at a model synapse in the chick ciliary ganglion, where ACh activates diffusely distributed α7- and α3-containing nAChRs (α7-nAChRs and α3*-nAChRs). Elevating quantal content lengthened EPSC decay time and prolonged both the fast (α7-nAChR-mediated) and slow (α3*-nAChR-mediated) components of decay, even in the presence of acetylcholinesterase. Increasing quantal content also prolonged decay times of pharmacologically isolated α7-nAChR- and α3*-nAChR-EPSCs. The effect upon EPSC decay time of changing quantal content was 5-10 times more pronounced for α3*-nAChR- than α7-nAChR-mediated currents and operated over a considerably longer time window: ≈ 20 vs ≈ 2 ms. Control experiments rule out a presynaptic source for the effect. We suggest that α3*-nAChR currents are prolonged at higher quantal content because of ACh spillover and postsynaptic potentiation (Hartzell et al., 1975), while α7-nAChR currents are prolonged probably for other reasons, e.g., increased occupancy of long channel open states. α3*-nAChRs report more spillover when α7-nAChRs are competitively blocked than under native conditions; this could be explained if α7-nAChRs buffer ACh and regulate its availability to activate α3*-nAChRs. Our results suggest that non-α7-nAChRs such as α3*-nAChRs may be suitable for paracrine nicotinic signaling but that α7-nAChRs may not be suitable. Our results further suggest that α7-nAChRs may buffer ACh and regulate its bioavailability.

Native α6β4∗ nicotinic receptors control exocytosis in human chromaffin cells of the adrenal gland

In the present study, we have electrophysiologically characterized native nicotinic acetylcholine receptors (nAChRs) in human chromaffin cells of the adrenal gland as well as their contribution to the exocytotic process. α-Conotoxin AuIB blocked by 14 ± 1% the acetylcholine (ACh)-induced nicotinic current. α-Conotoxin MII (α-Ctx MII) exhibited an almost full blockade of the nicotinic current at nanomolar concentrations (IC(50)=21.6 nM). The α6*-preferring α-Ctx MII mutant analogs, α-Ctx MII[H9A,L15A] and α-Ctx MII[S4A,E11A,L15A], blocked nAChR currents with an IC(50) of 217.8 and 33 nM, respectively. These data reveal that nAChRs in these cells include the α6* subtype. The washout of the blockade exerted by α-conotoxin BuIA (α-Ctx BuIA; 1 μM) on ACh-evoked currents was slight and slow, arguing in favor of the presence of a β4 subunit in the nAChR composition. Exocytosis was almost fully blocked by 1 μM α-Ctx MII, its mutant analogs, or α-Ctx BuIA. Finally, the fluorescent analog Alexa Fluor 546-BuIA showed distinct staining in these cells. Our results reveal that α6β4* nAChRs are expressed and contribute to exocytosis in human chromaffin cells of the adrenal gland, the main source of adrenaline under stressful situations.

Phenotype-specific down-regulation of nicotinic acetylcholine receptors in the pelvic ganglia of castrated rats: Implications for neurogenic erectile dysfunction

Pelvic ganglia (PG) play critical roles in relaying sympathetic and parasympathetic information from the spinal cord to the penile vasculature and, controlling the penile reflex. Animal studies have shown that androgen deprivation by castration causes erectile dysfunction (ED). Until now, however, neural mechanisms underlying castration-induced ED remain unclear. Therefore, we examined whether androgen deprivation down-regulates nicotinic acetylcholine receptors (nAchRs), which mediate fast excitatory synaptic transmission in the PG. Toward this end, neurogenic ED was demonstrated by measuring the intracavernous pressure in castrated rats. Real-time PCR analysis revealed that the transcripts encoding nAchR α3/α5/β4 subunits were significantly down-regulated in the PG neurons. In addition, down-regulation of the nAchR subunits was reversed by replacement of testosterone. Patch-clamp experiments showed that the nAchR currents were selectively attenuated in the parasympathetic PG neurons innervating the penile vasculature, activation of which elicits penile erection. Taken together, our data suggest that phenotype-specific down-regulation of nAchRs in the PG neurons may contribute to the neurogenic ED in castrated rats.

Molecular mechanisms of cross‐inhibition between nicotinic acetylcholine receptors and P2X receptors in myenteric neurons and HEK‐293 cells

P2X(2) and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and alpha3beta4subunit-containing nAChRs. The function of P2X2 and alpha3beta4 nAChRs expressed by HEK-293 cells and guinea pig ileum myenteric neurons in culture was studied using whole-cell patch clamp techniques. In HEK-293 cells expressing alpha3beta4 nAChRs and P2X2 receptors, co-application of ATP and acetylcholine caused inward currents that were 56 +/- 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells expressing alpha3beta4 nAChRs and a C-terminal tail truncated P2X2 receptor (P2X2TR) (P > 0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2 receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK-293 cells expressing both receptors. Constitutive inhibition did not occur in HEK-293 cells expressing alpha3beta4 nAChRs transfected with P2X2TR. Currents caused by low (< or =30 micromol L(-1)), but not high (> =100 micromol L(-1)) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co-expression with alpha3beta4 nAChRs. The C-terminal tail of P2X2 receptors mediates cross-inhibition between alpha3beta4 nAChR-P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross-inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co-transmitters.

Telithromycin blocks neuromuscular transmission and inhibits nAChR currents in vitro

Telithromycin, a ketolide antibiotic, is reported to exacerbate myasthenia gravis, potentially leading to respiratory failure and death. However, telithromycin is not associated with neuromuscular effects in animal toxicity studies. The objective of this study was to examine the effect of telithromycin on the neuromuscular junction in the isolated rat phrenic nerve-diaphragm preparation and to investigate its postsynaptic effects on the muscle-like nicotinic acetylcholine (ACh) receptors expressed on human TE671 cells. Telithromycin decreased the twitch contraction force of the rat diaphragm muscle in response to phrenic nerve stimulation in a concentration-dependent manner with an IC 50 of 22.3 μM and a maximal inhibition of ∼70%. The trans-membrane current from the ACh receptors expressed in the TE671 neuromedulloblastoma cells was recorded in the whole-cell patch-clamp configuration. When applied to the TE671 cells, telithromycin caused a dose-dependent inhibition of the nicotinic ACh current with an IC 50 of 3.5 μM and maximal inhibition of nearly 100%. These results indicate that telithromycin inhibits postsynaptic nicotinic ACh receptors in vitro and partially blocks neuromuscular transmission in the isolated rat phrenic nerve-diaphragm preparation. Based on these findings, we propose that exacerbation of myasthenia gravis reported in some patients taking telithromycin results in part from postsynaptic neuromuscular transmission block.

Calcineurin Suppression of Alpha7 nAChR Currents in Xenopus Oocytes

As part of our study of mechanisms underlying upregulation of neuronal nicotinic acetylcholine receptors (nAChRs) in brain, due to chronic nicotine exposure, we are characterizing signaling pathways that influence the magnitude of α7 nAChR currents in Xenopus oocytes. In previous studies of Kv1.1 channels in oocytes, we found that calcineurin (CaN) and protein tyrosine kinases (PTKs) mediated calcium-dependent suppression of Kv1.1 currents, through both endocytosis of channels as well as effects on channel gating. Thus, we hypothesized that these molecules would similarly regulate nAChR currents. We first determined that 100 uM bath-applied genistein (an inhibitor of many PTKs) enhanced murine peak α7 nAChR currents in oocytes, by a factor of 2-3, as reported by others (Cho et al., 2005, J. Neuroscience). We also measured an increase in membrane capacitance (Cm), as expected if genistein's potentiation of peak currents was due to increased addition of channels to the plasma membrane via exocytosis. Conversely, injection of vanadate ion (VO43- ) into oocytes expressing α7 nAChRs produced a dramatic reduction (∼90%) in peak currents, but no clear decrease in Cm. Exposure of oocytes expressing α7 nAChRs to 10 uM cyclosporine A (CsA), a potent and specific inhibitor of CaN, increased the magnitude of α7 currents by a factor of ∼2, and also increased Cm. The facilitatory effect of CsA upon α7 current was blocked by 100 uM H-7, a non-specific inhibitor of serine/threonine PKs. The combination of genistein and CsA produced a greater enhancement of the α7 current than either agent alone; their combined effect was approximately additive. Our results suggest that CaN activity may provide a readout of the intracellular calcium concentration, due to recent activity of α7 nAChRs, and thereby negatively regulate the number of α7 nAChRs expressed on the plasma membrane.

PACAP/PAC 1R signaling modulates acetylcholine release at neuronal nicotinic synapses

Neuropeptides collaborate with conventional neurotransmitters to regulate synaptic output. Pituitary adenylate cyclase-activating polypeptide (PACAP) co-localizes with acetylcholine in presynaptic nerve terminals, is released by stimulation, and enhances nicotinic acetylcholine receptor- (nAChR-) mediated responses. Such findings implicate PACAP in modulating nicotinic neurotransmission, but relevant synaptic mechanisms have not been explored. We show here that PACAP acts via selective high-affinity G-protein coupled receptors (PAC 1Rs) to enhance transmission at nicotinic synapses on parasympathetic ciliary ganglion (CG) neurons by rapidly and persistently increasing the frequency and amplitude of spontaneous, impulse-dependent nicotinic excitatory postsynaptic currents (sEPSCs). Of the canonical adenylate cyclase (AC) and phospholipase-C (PLC) transduction cascades stimulated by PACAP/PAC 1R signaling, only AC-generated signals are critical for synaptic modulation since the increases in sEPSC frequency and amplitude were mimicked by 8-Bromo-cAMP, blocked by inhibiting AC or cAMP-dependent protein kinase (PKA), and unaffected by inhibiting PLC. Despite its ability to increase agonist-induced nAChR currents, PACAP failed to influence nAChR-mediated impulse-independent miniature EPSC amplitudes (quantal size). Instead, evoked transmission assays reveal that PACAP/PAC 1R signaling increased quantal content, indicating that it modulates synaptic function by increasing vesicular ACh release from presynaptic terminals. Lastly, signals generated by the retrograde messenger, nitric oxide- (NO-) are critical for the synaptic modulation since the PACAP-induced increases in spontaneous EPSC frequency, amplitude and quantal content were mimicked by NO donor and absent after inhibiting NO synthase (NOS). These results indicate that PACAP/PAC 1R activation recruits AC-dependent signaling that stimulates NOS to increase NO production and control presynaptic transmitter output at neuronal nicotinic synapses.

Inhibition of Neuronal Nicotinic Acetylcholine Receptor Subtypes by α-Conotoxin GID and Analogues

alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

Neuronal Ca2+ sensor VILIP-1 leads to the upregulation of functional α4β2 nicotinic acetylcholine receptors in hippocampal neurons

The neuronal Ca2+-sensor protein VILIP-1, known to affect clathrin-dependent receptor trafficking, has been shown to interact with the cytoplasmic loop of the α4-subunit of the α4β2 nicotinic acetylcholine receptor (nAChR), which is the most abundant nAChR subtype with high-affinity for nicotine in the brain. The α4β2 nAChR is crucial for nicotine addiction and the beneficial effects of nicotine on cognition. Its dysfunction has been implicated in frontal lobe epilepsy, Alzheimer's disease and schizophrenia. Here we report that overexpression of VILIP-1 enhances ACh responsiveness, whereas siRNA against VILIP-1 reduces α4β2 nAChR currents of hippocampal neurons. The underlying molecular mechanism likely involves enhanced constitutive exocytosis of α4β2 nAChRs mediated by VILIP-1. The two interaction partners co-localize in a Ca2+-dependent manner with syntaxin-6, a Golgi-SNARE protein involved in trans-Golgi membrane trafficking. Thus, we speculate that regulation of VILIP-1-expression might modulate surface expression of ligand-gated ion channels, such as the α4β2 nAChRs, possibly comprising a novel form of physiological up-regulation of ligand-gated ion channels.

A Single in Vivo Application of Cholinesterase Inhibitors Has Neuron Type-Specific Effects on Nicotinic Receptor Activity in Guinea Pig Hippocampus

The present study was designed to test the hypothesis that an acute in vivo treatment with reversible or irreversible acetylcholinesterase (AChE) inhibitors modifies the activities of nicotinic receptors (nAChRs) in hippocampal neurons. Here, whole-cell nicotinic responses were recorded from CA1 interneurons in hippocampal slices obtained from male guinea pigs at 1, 7, or 14 days after treatment with the irreversible AChE inhibitor, soman (1x LD(50) s.c.), and/or the reversible AChE inhibitor, galantamine (8 mg/kg i.m.). Naive animals were used as controls. Three types of nAChR responses, namely types IA, II, and III, which were mediated by alpha 7, alpha 4 beta 2, and alpha 3 beta 2 beta 4 nAChRs, respectively, could be recorded from the interneurons. The magnitude of alpha 7 nAChR currents was neuron-type dependent. Stratum radiatum interneurons (SRIs) with thick initial dendrites had the largest alpha 7 nAChR currents. Acute challenge with soman caused sustained reduction of type IA current amplitudes recorded from stratum oriens interneurons and increased the ratio of acetylcholine- to choline-evoked current amplitudes recorded from SRIs. In guinea pigs that developed long-lasting convulsions after the soman challenge, there was a sustained reduction of alpha 3 beta 2 beta 4 nAChR responses. Acute treatment with galantamine had no effect on type IA or III responses, whereas it decreased the incidence of type II currents. Pretreatment of the guinea pigs with galantamine prevented the suppressive effect of soman on type III responses. The neuron type-specific changes in nAChR activity induced by soman, some of which could be prevented by galantamine, may contribute to the maintenance of pathological rhythms in the hippocampal neuronal network.