Phillips, Helen Jane; (1992) Studies of calcium channels in cultured neuroblastoma and muscle cells using monoclonal antibodies. Doctoral thesis (Ph.D.), Royal Free Hospital School of Medicine.

Text Studies_of_calcium_channels_in.pdf Download (4MB)

Abstract

The calcium channel from skeletal muscle is composed of five subunits: α1, α2, β, γ and σ. The α1, subunit contains the ion channel pore and a dihydropyridine binding site but a role for the α2 subunit is unclear. In this study the role of the α2 subunit in the functioning of the dihydropyridine-sensitive calcium channel has been investigated. More specifically, monoclonal antibodies raised against this subunit from rabbit skeletal muscle t-tubule membranes were used as probes to study the functioning of the channels using primarily electrophysiological methods. In order to study the action of the monoclonal antibodies on voltage-dependent calcium channels, whole-cell patch clamp recordings were made from neuroblastoma and muscle cell lines. In the mouse neuroblastoma x rat glioma hybrid (NG 108 15) cells at a holding potential of -40mV, two components of current were present: a dihydropyridine-sensitive current (L-type) and a dihydropyridine-insensitive current. At a holding potential of -80mV an additional transient (T-type) component was also present. After incubating the cells for 24 hours with the antibody, of the seven monoclonal antibodies tested, only one (mAb 11.14) had an effect on the whole cell currents. This monoclonal antibody selectively reduced the dihydropyridine-sensitive (L-type) component of current and decreased the time to peak of the current. However when it was applied acutely to the bath during whole-cell recording, it had no effects on the inward calcium channel currents. Potassium-stimulated 45calcium flux experiments confirmed the effect of mAb 11.14 after 24 hour incubation with the cells, by reducing the stimulated calcium flux whilst none of the other antibodies produced a significant effect. Monoclonal antibody 11.14 was also tested on a mouse muscle cell line (BC3H1). These cells had two components of calcium channel current. The major component was dihydropyridine-sensitive and had both slow activation and inactivation rates. The second dihydropyridine-insensitive component was much smaller and could be isolated by using a holding potential of -40mV. Of two antibodies (against the α2 subunit) tested after 24 hour incubation with the cells, again only mAb 11.14 had an effect. However, on these cells the antibody appeared to increase the rate of activation of the dihydropyridine-sensitive component without the corresponding reduction in amplitude. This effect was also observed when the antibody was applied acutely and suggests that the α2 subunit possibly has a role in directly modulating dihydropyridine-sensitive calcium channel function. The results presented from both cell types indicate that there must be antigenic differences between dihydropyridine-sensitive channels and the other channel types seen in these cells since the antibody only affected dihydropyridine-sensitive channels. In addition they indicate that the α2 subunit is present in NG 108 15 cells and must be in close association with the α1 subunit (where the pore is located) enabling it to affect the functioning of the channel.

Download activity - last month

Export as JSONXMLCSV

Download activity - last 12 months

Export as CSVXMLJSON

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item