Functional properties of ryanodine receptors in hippocampal neurons change during early differentiation in culture.
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid ryanodine (BODIPY-ryanodine) binding and Ca(2+) imaging were used to study the properties of ryanodine receptors (RyRs) and cytoplasmic Ca(2+) (Ca) changes in neurons cultured from the embryonic rat hippocampus during the earliest stages of differentiation. Baseline Ca levels declined from 164 +/- 5 (SD) nM at early stages to 70 +/- 4 nM in differentiated neurons. Fluorescent BODIPY-ryanodine binding signals identified activated RyRs in somata, which were eliminated by removal of external Ca(2+) or by blockage of Ca(2+) entry through L-type but not N-type Ca(2+) channels. The GABA synthesis inhibitor 3-mercaptopropionic acid completely abolished ryanodine binding. Caffeine or K(+)-depolarization inhibited the activity of RyRs at very early stages of differentiation but had stimulatory effects at later stages after a network of processes had formed. BayK-8644 stimulated RyRs throughout all regions of all differentiating cells. The results suggest that in differentiating embryonic hippocampal neurons the activity of RyRs is maintained via Ca(2+) entering through L-type Ca(2+) channels. The mode of activation of L-type voltage-gated Ca(2+) channels with either membrane depolarization or specific pharmacological agents affects the coupled activity of RyRs differently as neurons differentiate processes and networks.
Sukhareva
Manana
M
Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA. sukharem@ninds.nih.gov
Smith
Susan V
SV
Maric
Dragan
D
Barker
Jeffery L
JL
D015097:3-Mercaptopropionic Acid / Q000494:pharmacology; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012433:Ryanodine / Q000037:antagonists & inhibitors / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
D002120:Calcium Channel Agonists; D020746:Calcium Channels, L-Type; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D011189:Potassium Chloride; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D015097:3-Mercaptopropionic Acid; D002118:Calcium