Sleep-Related Hypermotor Epilepsy Associated Mutations Uncover Important Kinetic Roles Of Alpha 4 Beta 2-Nicotinic Acetylcholine Receptor Intracellular Structures

Sleep-related hypermotor epilepsy (SHE) is a group of seizure disorders prominently associated with mutations in nicotinic acetylcholine receptors (nAChR). The most prevalent central nervous system nAChR subtype contains alpha 4 and beta 2 subunits, in two ratios. (alpha 4 beta 2)(2)beta 2-nAChR have high agonist sensitivity (HS-isoform), whereas (alpha 4 beta 2)(2)alpha 4-nAChR agonist responses exhibit a small high-sensitivity, and a predominant low-sensitivity, phase of function (LS-isoform). Multiple non-synonymous mutations in the second and third transmembrane domains of alpha 4 and beta 2 subunits are associated with SHE. We recently demonstrated that two additional, SHE-associated, missense mutations in the major cytoplasmic loops of these subunits [alpha 4(R336H) and beta 2(V337G)] cause increased macroscopic function-per receptor. Here, we use single-channel patch-clamp electrophysiology to show that these mutations influence single-channel amplitudes and open- and closed-state kinetics. Pure populations of HS- or LS-isoform alpha 4 beta 2-nAChR were expressed by injecting either 1:10 or 30:1 alpha 4:beta 2 cRNA ratios, respectively, into Xenopus laevis oocytes. Functional properties of the resulting mutant alpha 4 beta 2-nAChR isoforms were compared to their wildtype counterparts. alpha 4(R336H) subunit incorporation minimally affected single-channel amplitudes, whereas beta 2(V337G) subunit incorporation reduced them significantly in both isoforms. However, for both mutant subunits, increased function-per-receptor was predominantly caused by altered single channel kinetics. The alpha 4(R336H) mutation primarily destabilizes desensitized states between openings. By contrast, the beta 2(V337G) mutation principally stabilizes receptor open states. The use of naturally-occurring and physiologically-impactful mutations has allowed us to define valuable new insights regarding the functional roles of nAChR intracellular domains. Further mechanistic context is provided by intracellular-domain structures recently published for other members of the Cys-loop receptor superfamily (alpha 3 beta 4-nAChR and 5-HT3AR).