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

作者：Weltzin Maegan M; Lindstrom Jon M; Lukas Ronald J; Whiteaker Paul^*

来源：Neuropharmacology, 2016, 102: 158-173.

DOI：10.1016/j.neuropharm.2015.11.004

• Nicotinic acetylcholine receptor
• Nocturnal frontal lobe epilepsy
• alpha 4 beta 2 isoform
• Cytoplasmic loop

Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in alpha 2, alpha 4 and beta 2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for beta 2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, alpha 4(R336H) and beta 2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on alpha 4 beta 2-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; (alpha 4)(2)(beta 2)(3)] or low-sensitivity [LS; (alpha 4)(3)((beta 2)(2)] via 1:10 or 30:1 [alpha 4:beta 2] cRNA injection ratios, respectively. An unbiased (1:1 [alpha 4:beta 2] cRNA) injection ratio was also used to study potential shifts in isoform expression. alpha 4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (I-max) in all preparations (140% increase compared to wild type control). beta 2(V337G)-containing receptors significantly increased I-max in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. alpha 4 beta 2-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 beta 2-subunit-specific [I-125]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the beta 2(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.

• 出版日期2016-3