Nicotine Normalizes Intracellular Subunit Stoichiometry of Nicotinic Receptors Carrying Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy

Abstract

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is linked with high penetrance to several distinct nicotinic receptor (nAChR) mutations. We studied (␣4) 3 (␤2) 2 versus (␣4) 2 (␤2) 3 subunit stoichiometry for five channel-lining M2 domain mutations: S247F, S252L, 776ins3 in ␣4, V287L, and V287M in ␤2. ␣4 and ␤2 subunits were constructed with all possible combinations of mutant and wild-type (WT) M2 regions, of cyan and yellow fluorescent protein, and of fluorescent and nonfluorescent M3-M4 loops. Sixteen fluorescent subunit combinations were expressed in N2a cells. Fö rster resonance energy transfer (FRET) was analyzed by donor recovery after acceptor photobleaching and by pixel-by-pixel sensitized emission, with confirmation by fluorescence intensity ratios. Because FRET efficiency is much greater for adjacent than for nonadjacent subunits and the ␣4 and ␤2 subunits occupy specific positions in nAChR pentamers, observed FRET efficiencies from (␣4) 3 (␤2) 2 carrying fluorescent ␣4 subunits were significantly higher than for (␣4) 2 (␤2) 3 ; the converse was found for fluorescent ␤2 subunits. All tested ADNFLE mutants produced 10 to 20% increments in the percentage of intracellular (␣4) 3 (␤2) 2 receptors compared with WT subunits. In contrast, 24-to 48-h nicotine (1 M) exposure increased the proportion of (␣4) 2 (␤2) 3 in WT receptors and also returned subunit stoichiometry to WT levels for ␣4S248F and ␤2V287L nAChRs. These observations may be relevant to the decreased seizure frequency in patients with ADNFLE who use tobacco products or nicotine patches. Fluorescence-based investigations of nAChR subunit stoichiometry may provide efficient drug discovery methods for nicotine addiction or for other disorders that result from dysregulated nAChRs.Nocturnal frontal lobe epilepsy (NFLE) is marked by seizures that include rhythmic and repetitive limb movements, rapid uncoordinated movements, dystonic posturing, complex motor activities such as sleep walking and pelvic thrusting, and the elevation of the trunk and head with ictal fear and vocalization. NFLE seizures occur primarily during phase 2 of non-rapid-eye-movement sleep. They rarely progress to tonic-clonic convulsions or status epilepticus. There are often uncertain distinctions between NFLE and paroxysmal sleep disorders. The "frontal" description arises from ictal electroencephalographic data, where available, and seizure semiology origin (Provini et al., 1999;Herman et al., 2001;Combi et al., 2004;Derry et al., 2006;Ryvlin et al., 2006).Autosomal dominant NFLE (ADNFLE) (Scheffer et al., 1995) is linked, with high penetrance, to at least six distinct nAChR mutations in ␣4␤2 neuronal nicotinic acetylcholine receptors (nAChRs) (Steinlein et al., 1997;Oldani et al., 1998;Combi et al., 2004;Wimmer et al., 2009). Three mutations are in the channel-lining M2 domain of the ␣4 subunit (S247F ϭ S6ЈF in the commonly used M2 domain renumbering for Cys-loop receptors, S252L ϭ S10ЈL, and 776ins3, after the 17Ј position), whereas two mutations occur in...

Insights into the Neurobiology of the Nicotinic Cholinergic System and Nicotine Addiction from Mice Expressing Nicotinic Receptors Harboring Gain-of-Function Mutations

“…After these studies, investigators created various knock-in mouse models by expressing the mouse equivalent to human ADNFLE mutations in ␣4 ( Klaassen et al, 2006;Teper et al, 2007) or ␤2 (Xu et al, 2010) nAChR genes. These models further support the notion that ADNFLE is mediated by mutant nAChRs, possibly as a result of augmented nAChR sensitivity (Rodrigues- , changes in ␣4␤2* nAChR stoichiometry (Son et al, 2009), or altered GABAergic network activity (Steinlein, 2010).…”

Pharmacological Chaperoning of Nicotinic Acetylcholine Receptors Reduces the Endoplasmic Reticulum Stress Response

“…It should be noted that, in most of our experiments, two or three ␣4 subunits per receptor contained a fluorescent protein label, which enabled us to identify nAChR-expressing Neuro-2a cells or neurons. Previous experiments with both cultured Neuro-2a cells and knock-in mice indicated that nAChRs containing such labeled ␣4 subunits were normal in every way studied (Nashmi et al, 2003(Nashmi et al, , 2007Fonck et al, 2009;Son et al, 2009). We cannot rule out the possibility that a large intracellular fluorophore in the ␣4 subunit can affect nAChR assembly, thereby enhancing an ER stress response.…”

“…General methods for pixel-by-pixel normalized Förster resonance energy transfer (NFRET) from sensitized acceptor emission were described previously Son et al, 2009;Srinivasan et al, 2011). For the present NFRET experiments, Neuro-2a cells were transfected with GalT-eCFP, ␣4-mcherry, and ␤2-eGFP subunit plasmids.…”

Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

“…␣4␤2*-nAChR are the most prevalent central nervous system (CNS) nAChR subtype, comprising Ϸ70% of all rodent CNS nAChR (2) and are implicated in a wide range of normal and pathological functions, including learning, memory, mood, and nicotine dependence among others (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). ␣4␤2*-nAChR functionally interact with nicotine at concentrations found in smokers and are the target of varenicline, currently the most successful smoking cessation pharmacotherapy (12,13).…”