The development of Ca2+ channel responses and their coupling to exocytosis in cultured cerebellar granule cells.
Using single-cell imaging, we investigated developmental changes in the modulation of KCl-evoked Ca2+ entry by various voltage-dependent Ca2+ channels and the coupling of these channels to exocytosis in cultured cerebellar granule neurons. A component of the KCl-evoked Ca2+ elevation sensitive to nifedipine and localized at cell somata, decreases with culture age. A component blocked by 200 nM omega-Agatoxin-IVA increases with age and whilst localized primarily at the cell somata, also becomes evident at the neurites. The change in activity between nifedipine-sensitive Ca2+ channels and omega-Agatoxin-IVA-sensitive Ca2+ channels occurs at 13 days in vitro at cell somata. A component of Ca2+ entry insensitive to nifedipine and 200 nM omega-Agatoxin-IVA is localized primarily at the neurites and is apparent at all ages. Single-cell imaging of exocytosis using FM1-43 destaining indicates that the residual, but not the nifedipine- or omega-Agatoxin-IVA-sensitive components of Ca2+ entry, modulates exocytosis. However cells cultured for 20-26 days develop a component of Ca2+ entry at the neurites which is sensitive to 200 nM omega-Agatoxin-IVA and omega-Conotoxin-MVIIC and which partially controls release. Immunolocalization studies reveal that binding sites for omega-Conotoxin-GVIA are present throughout development, even though this toxin does not inhibit KCl-evoked [Ca2+]c elevations or exocytosis. 300 nM omega-Agatoxin-IVA labels both somata and, at later developmental stages, neurites, consistent with the functional studies.
Harrold
J
J
Neurosciences Institute, Department of Pharmacology, University of Dundee, U.K.
Ritchie
J
J
Nicholls
D
D
Smith
W
W
Bowman
D
D
Pocock
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D005089:Exocytosis; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D007700:Kinetics; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009543:Nifedipine / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D013111:Spider Venoms / Q000378:metabolism / Q000494:pharmacology; D020867:omega-Agatoxin IVA; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D005456:Fluorescent Dyes; D010455:Peptides; D013111:Spider Venoms; D020867:omega-Agatoxin IVA; C049925:fura-2-am; D011189:Potassium Chloride; D020866:omega-Conotoxin GVIA; D009543:Nifedipine; D002118:Calcium; D016257:Fura-2