Deletion of the synaptic protein interaction site of the N-type (CaV2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells.
Presynaptic N-type Ca2+ channels (CaV2.2, alpha1B) 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 alpha1B 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 alpha1B Ca2+ channel subunit and transiently transfected either the wild-type alpha1B 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 alpha1B-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 (approximately 150 x 10(6) 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 alpha1B 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 alpha1B-containing Ca2+ channels to exocytosis.
Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, USA. harkinsa@slu.edu
Amy B
AB
Harkins
Anne L
AL
Cahill
James F
JF
Powers
Arthur S
AS
Tischler
Aaron P
AP
Fox
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020864:Calcium Channels, N-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D005089:Exocytosis; D051379:Mice; D008969:Molecular Sequence Data; D018377:Neurotransmitter Agents / Q000378:metabolism; D010673:Pheochromocytoma / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D017384:Sequence Deletion; D013569:Synapses / Q000378:metabolism; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000378:metabolism
D020864:Calcium Channels, N-Type; D018377:Neurotransmitter Agents; D011994:Recombinant Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins