Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Research Articles

Fourth Conference on Epileptogenesis, May 23–26, 2007, Pisa, Italy

Fourth Conference on Epileptogenesis, May 23–26, 2007, Pisa, Italy

Nocturnal epileptic seizures versus the arousal parasomnias

We review here the clinical features of Nocturnal Frontal Lobe Epilepsy (NFLE) and the arousal parasomnias, to define possible differential diagnostic criteria and their inter-observer reliability, and to determine whether both conditions share a common disturbance of arousal mechanisms. Polysomnographic recordings defined several types of seizures in NFLE: Paroxysmal Arousals (PA), Nocturnal Paroxysmal Dystonia (NPD), Epileptic Nocturnal Wanderings (ENW). There are however several difficulties with the diagnosis of NFLE, not the least being that EEG discharges during the seizures are not universally found. These diagnostic difficulties are highlighted, as well as the fact that studies of the inter-observer reliability of the video polysomnographic features of NFLE were found unsatisfactory, especially for the PA. When NFLE patients and their relatives were matched to control subjects and analysed in regard to personal and family history for the parasomnias, the arousal parasomnias were reported significantly higher by patients with NFLE and their relatives. This higher recurrence of a personal and family history for the parasomnias in NFLE patients suggests that the two conditions share a common underlying mechanism. Based on evidence from Autosomal Dominant NFLE, a genetic disorder with mutations in nicotinic acetylcholine receptors(nAchR), we hypothesise that arousal parasomnias and NFLE both, have abnormal arousal mechanisms in the dorsal cholinergic arousal branch triggering peculiar motor patterns in the medial frontal lobe regions.

Properties of mutated murine α4β2 nicotinic receptors linked to partial epilepsy

We characterized, by electrophysiological methods, two biophysical properties of murine recombinant alpha4beta2 nicotinic acetylcholine receptors (nAChR) bearing a mutation (alpha4:+L264alpha4:beta2 or alpha4:S252Falpha4:beta2) linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sensitivity to acetylcholine (ACh) was increased by the S252F substitution expressed in heterozygosis (alpha4:S252Falpha4:beta2) but was markedly reduced when this mutation was expressed in homozygosis (S252Falpha4:beta2). ACh sensitivity was not altered by the +L264 insertion. Moreover, receptor desensitization was significantly increased by both mutations expressed in heterozygosis. These results are in general agreement to those of rat and human recombinant receptors bearing the same mutations, thus contributing to validate the use of knock-in mice harboring ADNFLE mutations as models to study this pathology.

Nicotine-Induced Dystonic Arousal Complex in a Mouse Line Harboring a Human Autosomal-Dominant Nocturnal Frontal Lobe Epilepsy Mutation

We generated a mouse line harboring an autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) mutation: the alpha4 nicotinic receptor S248F knock-in strain. In this mouse, modest nicotine doses (1-2 mg/kg) elicit a novel behavior termed the dystonic arousal complex (DAC). The DAC includes stereotypical head movements, body jerking, and forelimb dystonia; these behaviors resemble some core features of ADNFLE. A marked Straub tail is an additional component of the DAC. Similar to attacks in ADNFLE, the DAC can be partially suppressed by the sodium channel blocker carbamazepine or by pre-exposure to a very low dose of nicotine (0.1 mg/kg). The DAC is centrally mediated, genetically highly penetrant, and, surprisingly, not associated with overt ictal electrical activity as assessed by (1) epidural or frontal lobe depth-electrode electroencephalography or (2) hippocampal c-fos-regulated gene expression. Heterozygous knock-in mice are partially protected from nicotine-induced seizures. The noncompetitive antagonist mecamylamine does not suppress the DAC, although it suppresses high-dose nicotine-induced wild-type-like seizures. Experiments on agonist-induced 86Rb+ and neurotransmitter efflux from synaptosomes and on alpha4S248Fbeta2 receptors expressed in oocytes confirm that the S248F mutation confers resistance to mecamylamine blockade. Genetic background, gender, and mutant gene expression levels modulate expression of the DAC phenotype in mice. The S248F mouse thus appears to provide a model for the paroxysmal dystonic element of ADNFLE semiology. Our model complements what is seen in other ADNFLE animal models. Together, these mice cover the spectrum of behavioral and electrographic events seen in the human condition.

Is Too Much Inhibition to Blame in Autosomal Dominant Nocturnal Frontal Lobe Epilepsy?

Seizures and Enhanced Cortical GABAergic Inhibition in Two Mouse Models of Human Autosomal Dominant Nocturnal Frontal Lobe Epilepsy. Klaassen A, Glykys J, Maguire J, Labarca C, Mody I, Boulter J. Proc Natl Acad Sci USA 2006;103(50):19152–19157. Selected mutations in the human α4 or β2 neuronal nicotinic acetylcholine receptor subunit genes cosegregate with a partial epilepsy syndrome known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To examine possible mechanisms underlying this inherited epilepsy, we engineered two ADNFLE mutations ( Chrna4S252F and Chrna4+L264) in mice. Heterozygous ADNFLE mutant mice show persistent, abnormal cortical electroencephalograms with prominent delta and theta frequencies, exhibit frequent spontaneous seizures, and show an increased sensitivity to the proconvulsant action of nicotine. Relative to WT, electrophysiological recordings from ADNFLE mouse layer II/III cortical pyramidal cells reveal a >20-fold increase in nicotine-evoked inhibitory postsynaptic currents with no effect on excitatory postsynaptic currents. i.p. injection of a subthreshold dose of picrotoxin, a use-dependent γ-aminobutyric acid receptor antagonist, reduces cortical electroencephalogram delta power and transiently inhibits spontaneous seizure activity in ADNFLE mutant mice. Our studies suggest that the mechanism underlying ADNFLE seizures may involve inhibitory synchronization of cortical networks via activation of mutant α4-containing nicotinic acetylcholine receptors located on the presynaptic terminals and somatodendritic compartments of cortical GABAergic interneurons.

The role of the nicotinic acetylcholine receptors in sleep-related epilepsy

The role of neuronal acetylcholine receptors (nAChRs) in epilepsy has been clearly established by the finding of mutations in a subset of genes coding for subunits of the nAChRs in a form of sleep-related epilepsy with familial occurrence in about 30% of probands and dominant inheritance, named autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sporadic and familial forms have similar clinical and EEG features. Seizures begin in middle childhood as clusters of sleep-related attacks with prominent motor activity, and sustained dystonic posturing. In addition to nocturnal seizures, psychosis or schizophrenia, behavioral disorders, memory deficits and mental retardation were described in some individuals. Although over hundred families are on record, only a minority of them have been linked to mutations in the genes coding for the α4, α2 and β2 ( CHRNA4, CHRNA2, and CHRNB2) subunits of the nAChRs, indicating that ADNFLE is genetically heterogeneous despite a relatively homogeneous clinical picture. Functional characterization of some mutations suggests that gain of the receptor function might be the basis for epileptogenesis. In vitro and in vivo studies have shown high density of nAChRs in the thalamus, over activated brainstem ascending cholinergic pathway and enhanced GABAergic function, reinforcing the hypothesis that cortico-subcortical networks, regulating arousal from sleep, play a central role in seizure precipitation in ADNFLE.

A major role of the nicotinic acetylcholine receptor gene CHRNA2 in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is unlikely

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is known to be caused by mutations in the transmembrane regions of the neuronal nicotinic acetylcholine receptor (nAChR) genes CHRNA4 and CHRNB2. A third nAChR gene, CHRNA2, has been recently reported as mutated in an Italian family with nocturnal frontal lobe epilepsy, nocturnal wandering and ictal fear. We have now evaluated the role of CHRNA2 in families with "classical" ADNFLE. Mutation screening was performed in 47 families by amplification and subsequent sequencing of part of CHRNA2 exon 6 containing transmembrane regions 1-3. Detected variants were tested in a case-control association study. No mutations were identified in the parts of CHRNA2 that contribute to the ion pore. Sequencing identified a novel synonymous nucleotide exchange (c.771C/T) that was also present in two of 78 controls and is therefore likely to be non-pathogenic. The absence of mutations in our sample of 47 families renders a major role of CHRNA2 in ADNFLE unlikely.

Further evidence of genetic heterogeneity in families with autosomal dominant nocturnal frontal lobe epilepsy

Purpose Mutations in the genes encoding the alfa 2, alfa 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor (nAChR) play a causative role in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Moreover, variations in the promoter of the corticotropic-releasing hormone gene ( CRH) were also associated with ADNFLE. Here, we investigated whether nine brain-expressed genes ( CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNB2, CHRNB3, CHRNB4), encoding distinct nAChR subunits, and CRH are associated with the disease in three distinct ADNFLE families from Southern Italy. Methods There were 14 living affected individuals (9 women), ranging in age from 14 to 57 years, pertaining to three unrelated families. Age at onset of seizures clustered around 9 years of age (range from 7 and 16 years, mean: 9.1 years ± 3.8). All affected individuals manifested nocturnal partial seizures of frontal lobe origin, which were well controlled by medications. Exon 5 of CHRNA4 and CHRNB2 genes, harboring all the known mutations, was sequenced in the probands. Then, we performed a linkage study on 13 affected and 26 non-affected individuals belonging to the three families with microsatellite markers and an intragenic polymorphisms encompassing the chromosome localization of the nAChR subunit genes and of the CRH gene. Results Mutational and linkage analyses allowed us to exclude the involvement of all known nAChR subunit genes and of the CRH gene in ADNFLE in our families. Conclusion Our results further illustrate the considerable genetic heterogeneity for such a syndrome, despite the quite homogeneous clinical picture. It is therefore reasonable to hypothesize that at least another gene not belonging to the nAChR gene family, in addition to CRH, is involved in the pathogenesis of ADNFLE.

Synthesis of Poison-Frog Alkaloids and Their Pharmacological Effects at Neuronal Nicotinic Acetylcholine Receptors

The flexible and efficient enantioselective synthesis of poison-frog alkaloids has been described using the highly stereoselective conjugate addition reactions as the key step. Several 5,8-disubstituted indolizidines and 1,4-disubstituted quinolizidines have been synthesized according to this strategy. Furthermore, 5,6,8-trisubstituted indolizidne type of poison-frog alkaloid 223A and unique tricyclic poison-frog alkaloid 205B have also been synthesized by sequential use of the above key conjugate addition reaction. Investigations of inhibitory effects of synthetic poison-frog alkaloids on neuronal nicotinic acetylcholine receptors have been conducted, and we found that most of the synthetic compounds showed inhibitory effects on the neuronal nicotinic acetylcholine receptors. Especially, the 5,8-disubstituted indolizidine 235B inhibited the α4 β2-neuronal nicotinic acetylcholine receptors in highly subtype-selective manner. These results suggested that the synthetic alkaloid 235B is a promising lead compound for the drugs designed to treat cholinergic disorders such as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).

Seizures and enhanced cortical GABAergic inhibition in two mouse models of human autosomal dominant nocturnal frontal lobe epilepsy

Selected mutations in the human alpha4 or beta2 neuronal nicotinic acetylcholine receptor subunit genes cosegregate with a partial epilepsy syndrome known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To examine possible mechanisms underlying this inherited epilepsy, we engineered two ADNFLE mutations (Chrna4(S252F) and Chrna4(+L264)) in mice. Heterozygous ADNFLE mutant mice show persistent, abnormal cortical electroencephalograms with prominent delta and theta frequencies, exhibit frequent spontaneous seizures, and show an increased sensitivity to the proconvulsant action of nicotine. Relative to WT, electrophysiological recordings from ADNFLE mouse layer II/III cortical pyramidal cells reveal a >20-fold increase in nicotine-evoked inhibitory postsynaptic currents with no effect on excitatory postsynaptic currents. i.p. injection of a subthreshold dose of picrotoxin, a use-dependent gamma-aminobutyric acid receptor antagonist, reduces cortical electroencephalogram delta power and transiently inhibits spontaneous seizure activity in ADNFLE mutant mice. Our studies suggest that the mechanism underlying ADNFLE seizures may involve inhibitory synchronization of cortical networks via activation of mutant alpha4-containing nicotinic acetylcholine receptors located on the presynaptic terminals and somatodendritic compartments of cortical GABAergic interneurons.

Role of genetics in the diagnosis and treatment of epilepsy

Epilepsy is a heterogeneous group of multifactorial diseases, the vast majority determined by interactions between many genes and environmental factors; however, there are rare epilepsy syndromes that can be caused by a single gene mutation and are inherited according to classical mendelian genetic principles. Finding disease-causing genetic mutations in epilepsy has provided new opportunities for aiding diagnosis and developing therapies. Thus, the discovery of KCNQ2 mutations in benign familial neonatal convulsions, SCN1A mutations in severe myoclonic epilepsy of infancy and in generalized epilepsy with febrile seizures plus, and CHRA4 and CHRB2 mutations in autosomal–dominant nocturnal frontal lobe epilepsy, has led to the establishment of epilepsy as a disorder of ion channel function and, furthermore, has led to the introduction of genetic tests that are available clinically to aid in diagnosis and treatment. At the present time, clinical use of genetic testing in epilepsy is greatest in suspected cases of severe myoclonic epilepsy of infancy, generalized epilepsy with febrile seizures plus, atypical cases of benign familial neonatal convulsions and ‘occult’ cases of autosomal–dominant nocturnal frontal lobe epilepsy without a family history. Overall, clinical use is limited by the low number of documented disease-associated mutations and the uncertain clinical significance of many test results. Further elucidation of the relationship between gene mutations and channel function will add value to genetic testing in the future, as will better characterization of the association between gene mutations and clinical phenotypes.

Tobacco habits modulate autosomal dominant nocturnal frontal lobe epilepsy

Mutations in neuronal nicotinic acetylcholine receptors have been demonstrated in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The beneficial effect of nicotine administration was previously reported in one single case. We investigated the influence of the tobacco habits of 22 subjects from two pedigrees with α4 mutations (776ins3 and S248F). Subjects were interviewed with respect to pattern of nicotine intake and seizures. Seizure freedom was significantly associated with tobacco use ( P = 0.024). All seven nonsmokers with manifest ADNFLE had persistent seizures. Seizure fluctuations, including long remissions, corresponded to changes in tobacco habits in several patients. One patient who recently had begun treatment with transdermal nicotine experienced improvement. We conclude that tobacco appears to be an environmental factor that influences seizure susceptibility in ADNFLE. Inactivation by desensitization of the mutant receptors by nicotine may explain the beneficial effect. The efficacy and safety of transdermal nicotine in ADNFLE should be further explored.

Alteration of the in vivo nicotinic receptor density in ADNFLE patients: a PET study

Nicotinic acetylcholine receptors (nAChRs) are involved in a familial form of frontal lobe epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In several ADNFLE families, mutations were identified in the nAChR alpha4 or beta2 subunit, which together compose the main cerebral nAChR. Electrophysiological assessment using in vitro expression systems indicated a gain of function of the mutant receptors. However the precise mechanisms by which they contribute to the pathogenesis of a focal epilepsy remain obscure, especially since alpha4beta2 nAChRs are known to be widely distributed within the entire brain. PET study using [18F]-F-A-85380, a high affinity agonist at the alpha4beta2 nAChRs, allows the determination of the regional distribution and density of the nAChRs in healthy volunteers and in ADNFLE patients, thus offering a unique opportunity to investigate some in vivo consequences of the molecular defect. We have assessed nAChR distribution in eight non-smoking ADNFLE patients (from five families) bearing an identified mutation in nAChRs and in seven age-matched non-smoking healthy volunteers using PET and [(18)F]-F-A-85380. Parametric images of volume of distribution (Vd) were generated as the ratio of tissue to plasma radioactivities. The images showed a clear difference in the pattern of the nAChR density in the brains of the patients compared to the healthy volunteers. Vd values revealed a significant increase (between 12 and 21%, P < 0.05) in the ADNFLE patients in the mesencephalon, the pons and the cerebellum when compared to control subjects. Statistical parametric mapping (SPM) was then used to better analyse subtle regional differences. This analysis confirmed clear regional differences between patients and controls: patients had increased nAChR density in the epithalamus, ventral mesencephalon and cerebellum, but decreased nAChR density in the right dorsolateral prefrontal region. In five patients who underwent an additional [(18)F]-fluorodeoxyglucose (FDG) PET experiment, hypometabolism was observed in the neighbouring area of the right orbitofrontal cortex. The demonstration of a regional nAChR density decrease in the prefrontal cortex, despite the known distribution of these receptors throughout the cerebral cortex, is consistent with a focal epilepsy involving the frontal lobe. We also propose that the nAChR density increase in mesencephalon is involved in the pathophysiology of ADNFLE through the role of brainstem ascending cholinergic systems in arousal.

Synthesis of Poison Frog Alkaloids : Novel, Potent and Subtype-selective Blockers of Neuronal Nicotinic Acetylcholine Receptors

This article describes the synthesis of poison frog alkaloids using the highly stereoselective conjugate addition reaction as the key step. First total synthesis of the alkaloid 223A has been achieved. The proposed structure for natural 223A was revised and the relative stereostructure was determined by this total synthesis. First total syntheses of tricyclic alkaloid 205B and quinolizididne 207I have been achieved, and the absolute stereochemistry of both natural products was determined by these total syntheses. Investigations of the inhibitory effects of synthetic poison frog alkaloids on neuronal nicotinic acetylcholine receptors have also been conducted, and we found the 5, 8-disubstituted indolizidine 235B'is a promising lead compound for the drug design to treat autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).

Novel Seizure Phenotype and Sleep Disruptions in Knock-In Mice with Hypersensitive α4*Nicotinic Receptors

A leucine to alanine substitution (L9'A) was introduced in the M2 region of the mouse alpha4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, alpha4(L9'A)beta2 nAChRs were > or =30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9'A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on alpha4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of alpha4or beta2 subunits. Thalamic cultures and synaptosomes from L9'A mice were hypersensitive to nicotine-induced ion flux. L9'A mice were approximately 15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9'A mice differed qualitatively from those in WT: L9'A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9'A mice were partial, whereas WT seizures were generalized. When L9'A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure approximately 20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9'A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive alpha4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE.

Genetic Focal Epilepsies: State of the Art and Paths to the Future

The concept of genetic focal epilepsies is relatively new as compared to awareness of the importance of genetic factors in the generalized epilepsies. However, in the past decade, there has been increasing recognition of families with dominantly inherited partial epilepsies. Better definition of the phenotypes allows identification of distinct syndromes. The main familial focal epilepsies are autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE), familial mesial TLE (FMTLE), familial lateral TLE (FLTLE), and familial partial epilepsy with variable foci (FPEVF). The only genes identified so far are those for ADNFLE and FLTLE. In these disorders, functional studies are the next step and could provide advances leading to clarification of the pathophysiology as well as to new therapeutic strategies. At present, we can provide genetic counseling and a more accurate prognosis for most of the familial focal epilepsies. Greater awareness of the genetic basis in this group of disorders by the treating physicians is essential for identification of new families. This will allow further linkage studies, candidate gene screening, and identification of new genes, which will hopefully result in genetically based prevention and treatment.

Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human α4β2 Acetylcholine Receptors

Human neuronal nicotinic acetylcholine receptor (AChR) alpha4 subunits and an alpha4 mutant (S247Falpha4) found in autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) were expressed along with beta2 in permanently transfected tsA201 human embryonic kidney cell lines. Their sensitivity to activation, desensitization, and up-regulation by cholinergic ligands was investigated. Up-regulation after 3 to 24 h resulted primarily from an increase in assembly of AChRs from large pools of unassembled subunits, but up-regulation also resulted from a 5-fold increase in the lifetime of AChRs in the surface membrane. Up-regulation does not require current flow through surface membrane AChRs, because up-regulation occurs in the presence of the channel blocker mecamylamine and with the alpha4 mutant, which prevents nearly all AChR function. Both membrane-permeable ligands like nicotine and much less permeable quaternary amine cholinergic ligands can act as pharmacological chaperones within the endoplasmatic reticulum to promote the assembly of AChRs. Agonists are more potent pharmacological chaperones than antagonists, presumably because activated or desensitized conformations assemble more efficiently. Assembly intermediates are disrupted by solubilization in Triton X-100, but chemical cross-linking stabilizes a putative assembly intermediate approximately the size of an alpha4beta2alpha4beta2 tetramer.

Two new putative susceptibility loci for ADNFLE

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) has been up to now considered a simple Mendelian trait caused by mutations in neuronal nicotinic acetylcholine receptor (nAChR) subunit genes. We previously demonstrated that in a three-generation Italian family the disease was unlinked to all known ADNFLE loci as well as to all known brain-expressed nAChR subunits. The genome-wide linkage analysis here presented performed on this family points to the existence of two new putative ADNFLE loci on chromosomes 3p22-p24 and 8q11.2-q21.1. These findings, together with several ADNFLE characteristics, suggest that this epilepsy could be, at least in the above family, a complex disorder. In particular, we propose and discuss the hypothesis of a digenic transmission of the disease.

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca2+ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Extracellular Ca(2+) robustly potentiates the acetylcholine response of alpha4beta2 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-alpha4(S252F), alpha4(S256L), alpha4(+L264), beta2(V262L), and beta2(V262M)-reduced 2 mM Ca(2+) potentiation of the alpha4beta2 1 mM acetylcholine response by 55 to 74%. To determine whether altered allosteric Ca(2+) activation or enhanced Ca(2+) block caused this reduction, we coexpressed the rat ADNFLE mutations with an alpha4 N-terminal mutation, alpha4(E180Q), that abolished alpha4beta2 allosteric Ca(2+) activation. In each case, Ca(2+) inhibition of the double mutants was less than that expected from a Ca(2+) blocking mechanism. In fact, the effects of Ca(2+) on the ADNFLE mutations near the intracellular end of the M2 region-alpha4(S252F) and alpha4(S256L)-were consistent with a straightforward allosteric mechanism. In contrast, the effects of Ca(2+) on the ADNFLE mutations near the extracellular end of the M2 region-alpha4(+L264)beta2, beta2(V262L), and beta2(V262M)-were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca(2+) on the alpha4beta2, alpha4(+L264)beta2, and alpha4beta2(V262L) single-channel conductances, the effects of membrane potential on the beta2(V262L)-mediated reduction in Ca(2+) potentiation, and the effects of eliminating the negative charges in the extracellular ring on this reduction failed to provide any direct evidence of mutant-enhanced Ca(2+) block. Moreover, analyses of the alpha4beta2, alpha4(S256L), and alpha4(+L264) Ca(2+) concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca(2+) potentiation of the alpha4beta2 acetylcholine response by altering allosteric activation rather than by enhancing block.

Symposium II

Symposium II