Distinctive effects of nicotinic receptor intracellular-loop mutations associated with nocturnal frontal lobe epilepsy

Abstract

Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in α2, α4 and β2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for β2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, α4(R336H) and β2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on α4β2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations’ effects at alternate isoforms: high-sensitivity [HS; (α4)2(β2)3] or low-sensitivity [LS; (α4)3(β2)2] via 1:10 or 30:1 [α4:β2] cRNA injection ratios, respectively. An unbiased (1:1 [α4:β2] cRNA) injection ratio was also used to study potential shifts in isoform expression. α4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). β2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. α4β2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using β2-subunit-specific [125I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the β2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between TM- and C2- domain AD/NFLE-associated mutations. As we discuss, the shared features may be particularly salient to AD/NFLE etiology.

Rationale for an adjunctive therapy with fenofibrate in pharmacoresistant nocturnal frontal lobe epilepsy

“…Five subjects reported a family history of epilepsy (ADNFLE) with confirmed mutation at genetic test (PZ 6 CHRNA4 mut and PZ 7‐10 CHRNA2). Due to the well‐established genetic heterogeneity despite a relatively homogeneous clinical picture, genetic testing in our sporadic cases was not regarded as relevant, especially after a cost–benefit analysis . Seven subjects described parasomnias or nocturnal motor events in first‐grade relatives.…”

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

“…mAb 295 is a monoclonal antibody that specifically recognizes correctly folded human, bovine, and rodent nAChR ␤2 subunits (41)(42)(43). The protocol used has previously been described (26,36,44). In brief, following determination of expressed nAChR function by maximal stimulation (I max , where I max is peak current response) with ACh (EC 100 , 1 s), sets of six oocytes each expressing individual concatemeric ␣4␤2-nAChR isoforms or variants thereof were sorted into a 24-well plate (one set per well).…”

“…The expression of ␣4␤2-nAChR isoforms appears to be physiologically significant. For example, multiple epilepsy-associated ␣4 and ␤2 subunit mutants alter ratios of HS to LS ␣4␤2-nAChR isoforms (17,26), and agonists capable of preferentially stimulating LS ␣4␤2-nAChR produce distinctive physiological effects (27)(28)(29). Accordingly, a better understanding of the respective roles of HS and LS ␣4␤2*-nAChR isoforms is likely to have considerable translational implications.…”

Probing Binding Interactions of Cytisine Derivatives to the α4β2 Nicotinic Acetylcholine Receptor

“…Changes in subunit stoichiometry are linked to both nicotine addiction and autosomal dominant nocturnal frontal lobe epilepsy. 7,[12][13][14] Therefore, there is strong motivation for gaining a better understanding of differential agonist effects on these two stoichiometries of the α4β2 nAChR. Figure 1.…”