Rare missense variants of neuronal nicotinic acetylcholine receptor altering receptor function are associated with sporadic amyotrophic lateral sclerosis.

Sporadic amyotrophic lateral sclerosis (SALS) is a motor neuron degenerative disease of unknown etiology. Current thinking on SALS is that multiple genetic and environmental factors contribute to disease liability. Since neuronal acetylcholine receptors (nAChRs) are part of the glutamatergic pathway, we searched for sequence variants in CHRNA3, CHRNA4 and CHRNB4 genes, encoding neuronal nicotinic AChR subunits, in 245 SALS patients and in 450 controls. We characterized missense variants by in vitro mutagenesis, cell transfection and electrophysiology. Sequencing the regions encoding the intracellular loop of AChRs subunits disclosed 15 missense variants (6.1%) in 14 patients compared with only six variants (1.3%) in controls (P = 0.001; OR 4.48, 95% CI 1.7-11.8). The frequency of variants in exons encoding extracellular and transmembrane domains and in intronic regions did not differ. NAChRs formed by mutant alpha3 and alpha4 and wild-type (WT) beta4 subunits exhibited altered affinity for nicotine (Nic), reduced use-dependent rundown of Nic-activated currents (I(Nic)) and reduced desensitization leading to sustained intracellular Ca(2+) concentration, in comparison with WT-nAChR. The cellular loop has a crucial importance for receptor trafficking and regulating ion channel properties. Missense variants in this domain are significantly over-represented in SALS patients and alter functional properties of nAChR in vitro, resulting in increased Ca(2+) entry into the cells. We suggest that these gain-of-function variants might contribute to disease liability in a subset of SALS because Ca(2+) signals mediate nAChR's neuromodulatory effects, including regulation of glutamate release and control of cell survival.

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

The epsilon subunit of the human endplate ACh receptor (AChR) is a key determinant of the large fraction of the ACh-evoked current carried by Ca2+ ions (P(f)). Consequently, missense mutations in the epsilon subunit are potential targets for altering the P(f) of human AChR. In this paper we investigate the effects of two pathogenic point mutations in the M2 transmembrane segment AChR epsilon subunit, epsilonT264P and epsilonV259F, that cause slow-channel syndromes (SCS). When expressed in GH4C1 cells, the mutant receptors subunits raise Ca2+ permeability of the receptors approximately 1.5 and approximately 2-fold above that of wild-type, to attain P(f) values of 11.8% (epsilonT264P) and 15.4% (epsilonV259F). The latter value exceeds most P(f) values reported to date for ligand-gated ion channels. Consistent with these findings, the biionic Ca2+ permeability ratio (P(Ca)/P(Cs)) of the mutant AChRs is also increased. Upon repetitive stimulation with ACh, the mutant receptors show an enhanced current run-down compared with wild-type, leading to a strong reduction of their function. We propose that the enhanced Ca2+ permeability of the mutant receptors overrides the protective effect of desensitization and, together with the prolonged opening events of the AChR channel, is an important determinant of the excitotoxic endplate damage in the SCS.

Modulation of sensory neuron mechanotransduction by PKC- and nerve growth factor-dependent pathways.

Many sensations of pain are evoked by mechanical stimuli, and in inflammatory conditions, sensitivity to such stimuli is commonly increased. Here we used cultured sensory neurons as a model of the peripheral terminal to investigate the effects of inflammatory signaling pathways on mechanosensitive ion channels. Activation of two of these pathways enhanced transduction in a major population of nociceptors. The proinflammatory neurotrophin nerve growth factor caused an up-regulation of mechanically activated currents via a transcriptional mechanism. Activators of PKC, given in vitro and in vivo, also caused an increase in mechanically activated membrane current and behavioral sensitization to mechanical stimulation, respectively. The effect of activating PKC was inhibited by tetanus toxin, suggesting that insertion of new channels into the cell membrane is involved in sensitization. These results reveal previously undescribed mechanisms by which PKC and nerve growth factor synergistically enhance the response of nociceptors to mechanical stimuli, suggesting possible targets for pain treatment.