Novel β subunit mutation causes a slow-channel syndrome by enhancing activation and decreasing the rate of agonist dissociation

Abstract

We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V --> F substitution in the M1 domain of the beta subunit (betaV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation betaV229F expressed normal amounts of AChRs and decreased the ACh EC50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the betaV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the betaV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the betaV229F mutation on the miniature endplate currents and thus are direct evidence that the betaV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.

Functional contribution of α3L8′ to the neuronal nicotinic α3 receptor

“…The results of mutating position L8′ in the α3 nAChR contrast quite dramatically with those obtained for the SCS mutation βV229F, for which this study was, in part, designed (Navedo et al, 2006). Although the βV229F mutation produced a 10‐fold decrease in ACh EC 50 , the α3 mutant receptors showed higher or unaffected EC 50 s. Our results suggest that this disparity may be explained based on differences in the local environment of the studied position between α and β subunits (Akabas and Karlin, 1995) and in subunit composition between neuronal and muscle nAChRs.…”

“…We characterized the contribution of position α3L8′, a conserved residue in TMD1, to the functional properties of α3β2 and α3β4 neuronal nicotinic receptor subtypes. L8′ is homologous to position V229 in TMD1 of the muscle β subunit, in which a natural substitution of Phe for the WT Val causes CMS (Vohra et al, 2004; Navedo et al, 2006). The high degree of amino acid conservation and the disruption of receptor function caused by the spontaneous mutation in the homologous position in muscle‐type receptors suggested a role of L8′ in ion channel function.…”

“…Previous reports have shown that mutations at the amino terminal portion of muscle α and β subunits' TMD1 allosterically affect agonist binding (Wang et al, 1997; Navedo et al, 2006), although effects on channel gating are also possible. These spontaneous mutations occurred at the same position studied in this work (βV229F) or one position pre‐N‐terminal to it (αN217K).…”

“…1). Second, a couple of naturally occurring mutations in the muscle‐type AChR, shown to cause slow‐channel syndrome (SCS), have been identified to occur at this position (βV229F; Navedo et al, 2006) or one position more N‐terminal to it (αN217K; Wang et al, 1997). Albeit by different mechanisms, these two SCS mutations were shown to affect agonist binding, implying that this region may play an important role in coupling agonist binding to channel gating either allosterically or through the so‐called pre‐M1 region (Lee and Sine, 2005).…”

Pathogenic point mutations in a transmembrane domain of the ε subunit increase the Ca2+ permeability of the human endplate ACh receptor

“…The enhanced Ca 2+ permeability of the ε SCS mutants additionally increases the Ca 2+ ingress into the junctional sarcoplasm that stems from the prolonged duration of the synaptic current and the intrinsically high Ca 2+ permeability of human AChR and is thus directly relevant to the pathogenesis of the endplate myopathy associated with the observed mutations. An increased permeability to Ca 2+ may also occur with SCS mutations in other AChR subunits, as an increase of P Ca / P Cs was observed in HEK 293 cells expressing mouse βV299F‐AChR, a mutant receptor designed after a recently identified mutation in a SCS patient (Navedo et al 2006). Both εV259F and εT264P mutations yield a complex effect on receptor function.…”