Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type skeletal muscle voltage-gated calcium channels (VGCCs)
MATERIALS AND METHODSCell Culture. Parental embryonic day 15 rat ventral spinal neuron preparation, mouse N18TG2 neuroblastoma culture, somatic cell fusion, and hybrid selection were performed as described (25,26). VSC 4.1 hybrid cells were maintained in logarithmic-phase growth on poly(L-ornithine)-precoated T-75 flasks (Coming) in Dulbecco's modified Eagle's medium/F-12 growth medium (GIBCO) containing Sato's components (Sigma) and 2% heat inactivated newborn calf serum (HyClone). VSC 4.1 cells undergoing cAMP-induced differentiation were plated at 1.2-1.5 x 106 cells per 75-mm2 flask and treated for 7 days with 1 mM dibutyryl cAMP or 1 mM 8-bromo-cAMP before use. Aphidicolin (0.4 pg/ml) (27) (Sigma) was added for 2 days after 24 hr in cAMP, and after 2 additional days of aphidicolin-free growth surviving cells were maintained in medium containing 1 mM cAMP and 0.025-0.05 ,ug of aphidicolin per ml. Differentiated hybrids were harvested by trituration after 1-hr incubation at 370C in Ca2+/Mg2+-free Hanks' balanced salt solution containing 1 mM EDTA and transferred into 24-well plates (Coming) at 2.0 x 104 cells per well [for direct cell count and lactate dehydrogenase (LDH) assays] or per 11-mm-diameter glass coverslip (for fluorescence assays). Pharmacologic agents and immunoglobulins were added to cells 24 hr after replating and were maintained in growth medium for the entire experAbbreviations: ALS, amyotrophic lateral sclerosis; LEMS, Lam-bert-Eaton myasthenic syndrome; VGCC, voltage-gated calcium channel; FDA, fluorescein diacetate; PI, propidium iodide; LDH, lactate dehydrogenase; AMPA, DL-a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.tPresent address: