The isolation and characterisation of the genes coding for the calcium channel affected by Lambert-Eaton myasthenic syndrome antibodies in NG108-15 cells

Thesis

Lambert-Eaton myasthenic syndrome is a rare paraneoplastic and autoimmune disorder affecting the presynaptic voltage gated calcium channels at the neuromuscular junction. The aim of this project was to isolate the genes coding for the α1 and α2 subunits of the voltage-gated calcium channel from hybrid neuronal cell lines including NG108-15 whose potassium stimulated ⁴⁵Ca²⁺ flux is reduced by IgG from patients with Lambert-Eaton myasthenic syndrome.

The cDNA libraries were constructed in λgt10 from NG108-15 mRNA and screened with PCR products derived from the rabbit skeletal muscle α1 and α2-δ genes. A 2230 bp portion of the NG108-15 α2-δ gene homologous to bp 1000- 3304 of the rabbit gene has been isolated and sequenced. This shows 48% homology to the amino acid sequence of the rabbit gene and 56% nucleotide homology with particular conservation of the C-tenninal domains.

The NG108-l5 α2 mRNA is 8.5 kb long and was found in the N18TG2 mouse neuroblastoma cell line from which NG108-15 is derived and in mouse brain. Rabbit α2-δ gene PCR primers were used to screen a rodent tissue cDNA panel to detect highly homologous sequences by PCR amplification. These products were detected only in mouse brain and lung and in rat skeletal muscle. The mouse brain and lung product amino acid sequences were 87% homologous to the rabbit gene while the rat skeletal muscle product was 81% homologous and both differed significantly from the homologous region of the NG108-15 gene.

This suggests that some of the diversity of voltage gated calcium channels arises from the expression of different α2-δ subunits within the 5 subunit VGCC complex as well as from diversity in the other subunits.

Authors: von Rosch, A

• Peer review status: Peer reviewed
• DOI: 10.5287/ora-0ng0pbkom
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford