Measurement of intracellular free zinc in living cortical neurons: routes of entry.
We used the ratioable fluorescent dye mag-fura-5 to measure intracellular free Zn2+ ([Zn2+]i) in cultured neocortical neurons exposed to neurotoxic concentrations of Zn2+ in concert with depolarization or glutamate receptor activation and identified four routes of Zn2+ entry. Neurons exposed to extracellular Zn2+ plus high K+ responded with a peak cell body signal corresponding to a [Zn2+]i of 35-45 nM. This increase in [Zn2+]i was attenuated by concurrent addition of Gd3+, verapamil, omega-conotoxin GVIA, or nimodipine, consistent with Zn2+ entry through voltage-gated Ca2+channels. Furthermore, under conditions favoring reverse operation of the Na+-Ca2+ exchanger, Zn2+ application induced a slow increase in [Zn2+]i and outward whole-cell current sensitive to benzamil-amiloride. Thus, a second route of Zn2+ entry into neurons may be via transporter-mediated exchange with intracellular Na+. Both NMDA and kainate also induced rapid increases in neuronal [Zn2+]i. The NMDA-induced increase was only partly sensitive to Gd3+ or to removal of extracellular Na+, consistent with a third route of entry directly through NMDA receptor-gated channels. The kainate-induced increase was highly sensitive to Gd3+ or Na+ removal in most neurons but insensitive in a minority subpopulation ("cobalt-positive cells"), suggesting that a fourth route of neuronal Zn2+ entry is through the Ca2+-permeable channels gated by certain subtypes of AMPA or kainate receptors.
Sensi
S L
SL
Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Canzoniero
L M
LM
Yu
S P
SP
Ying
H S
HS
Koh
J Y
JY
Kerchner
G A
GA
Choi
D W
DW
D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D000001:Calcimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology; D002478:Cells, Cultured; D004558:Electric Stimulation; D004594:Electrophysiology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D007476:Ionophores / Q000494:pharmacology; D007608:Kainic Acid / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D016202:N-Methylaspartate / Q000494:pharmacology; D019579:Neocortex / Q000166:cytology; D009474:Neurons / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*; D009553:Nimodipine / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D015032:Zinc / Q000032:analysis* / Q000378:metabolism*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D005456:Fluorescent Dyes; D007476:Ionophores; D011810:Quinoxalines; D019831:Sodium-Calcium Exchanger; C094537:mag-Fura 5; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; D000001:Calcimycin; D009553:Nimodipine; D016202:N-Methylaspartate; C033170:4-bromo-A-23187; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D008274:Magnesium; D015032:Zinc; D007608:Kainic Acid; D002118:Calcium; D016257:Fura-2