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 ?2, ?4 and ?2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for ?2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, ?4(R336H) and ?2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on ?4?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; (?4)2(?2)3] or low-sensitivity [LS; (?4)3(?2)2] via 1:10 or 30:1 [?4:?2] cRNA injection ratios, respectively. An unbiased (1:1 [?4:?2] cRNA) injection ratio was also used to study potential shifts in isoform expression. ?4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). ?2(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. ?4?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 ?2-subunit-specific [(125)I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the ?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.

SUBMITTER: Weltzin MM 

PROVIDER: S-EPMC4698238 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature