Metabolism of omega-conotoxin-sensitive voltage-operated calcium channels in human neuroblastoma cells: modulation by cell differentiation and anti-channel antibodies.
The turnover of voltage-operated calcium channels was studied in two different human neuroblastoma cell lines (IMR32 and SH-SY5Y) using omega-conotoxin. The 125I-omega-conotoxin bound to surface channels was internalized and degraded by the cells in a time- and temperature-dependent manner. The radioactive degradation products released in the medium were all trichloroacetic acid soluble and no longer recognized by anti-omega-conotoxin antibodies. Altering the pH of intracellular organelles with chloroquine and inhibiting lysosomal proteases with leupeptin reduced 125I-omega-conotoxin degradation but had no effect on its internalization. Postlabeling measurements showed that the rates of 125I-omega-conotoxin internalization and degradation were equal to the rate of channel removal from the cell surface after protein synthesis inhibition. The rate of removal of omega-conotoxin binding sites was parallel to the rate of loss of functional channels, as measured by means of the fura-2 technique. Drug-induced differentiation of human neuroblastoma cells slowed down channel internalization and degradation rates, leading to the known increased expression of plasma membrane calcium channels in differentiated cells. On the other hand, both human (from Lambert-Eaton myasthenic patients) and murine (from immunized mice) anti-channel antibodies increased the rates of channel internalization and degradation, leading to channel downregulation. The activity of presynaptic calcium channels is already known to be acutely modulated by a number of different agents (e.g., hormones and neurotransmitters); our studies suggest that a different form of channel modulation (changes in the number of channels due to interference with channel turnover) may be active over a longer time scale in neurons.(ABSTRACT TRUNCATED AT 250 WORDS)
Passafaro
M
M
Department of Medical Pharmacology, University of Milan, Italy.
Clementi
F
F
Sher
E
E
D000906:Antibodies / Q000276:immunology* / Q000502:physiology; D015220:Calcium Channels / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D002454:Cell Differentiation; D002738:Chloroquine / Q000494:pharmacology; D004594:Electrophysiology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D010456:Peptides, Cyclic / Q000494:pharmacology*; D013696:Temperature; D020960:omega-Conotoxins
D000906:Antibodies; D015220:Calcium Channels; D007976:Leupeptins; D010456:Peptides, Cyclic; D020960:omega-Conotoxins; C053423:Conus magus toxin; D002738:Chloroquine; C032854:leupeptin