Deletion of the Synaptic Protein Interaction Site of the N-Type ( Ca V2.2) Calcium Channel Inhibits Secretion in Mouse Pheochromocytoma Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS 2004-10, Vol.101 (42), p.15219-15224
• Main Authors: Harkins, Amy B., Cahill, Anne L., Powers, James F., Tischler, Arthur S., Fox, Aaron P., Catterall, William A.
• Format: Article
• Language: English
• Subjects: Albs; Animals; Binding sites; Binding Sites - genetics; Biological Sciences; Calcium; Calcium Channels, N-Type - chemistry; Calcium Channels, N-Type - genetics; Calcium Channels, N-Type - metabolism; Calcium Signaling; Capacitance; Cell Line, Tumor; Cell lines; Cells; Chromaffin cells; Exocytosis; Mice; Molecular Sequence Data; Neurons; Neurotransmitter Agents - secretion; Neurotransmitters; PC12 cells; Pheochromocytoma - genetics; Pheochromocytoma - metabolism; Pheochromocytoma - secretion; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rodents; Secretion; Sequence Deletion; SNARE Proteins; Synapses - metabolism; Transfection; Vesicular Transport Proteins - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0401001101

Presynaptic N-type Ca2+ channels ( Ca v2.2, α 1 B) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the α 1 B subunit. Whether binding of syntaxin to the N-type Ca2+ channels is required for coupling Ca2+ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, we deleted the synprint site from a recombinant α 1 B Ca2+ channel subunit and transiently transfected either the wild-type α 1 B or the synprint deletion mutant into mouse pheochromocytoma (MPC) cell line 9/3L, a cell line that has the machinery required for rapid stimulated exocytosis but lacks endogenous voltage-dependent Ca2+ channels. Secretion was elicited by activation of exogenously transfected Ca2+ channel subunits. The current-voltage relationship was similar for the wild-type and mutant α 1 B-containing Ca2+ channels. Although total Ca2+ entry was slightly larger for the synprint deletion channel, compared with the wild-type channel, when Ca2+ entry was normalized to cell size and limited to cells with similar Ca2+ entry (≈ 150× 106 Ca2+ ions/pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels. Furthermore, the amount of endocytosis was significantly reduced in cells with the α 1 B synprint deletion mutant, compared with the wild-type subunit. These results suggest that the synprint site is necessary for efficient coupling of Ca2+ influx through α 1 B-containing Ca2+ channels to exocytosis.