Single-channel activity in cultured cortical neurons of the rat in the presence of a toxic dose of glutamate.
Rat cortical neurons grown in cell culture were exposed to 500 microM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca(2+-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K+ channel requiring intracellular calcium ([Ca2+]i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 +/- 6 pS; 'BK channel'). Another Ca(2+)-dependent channel permeable for Cl- (conductance for outward currents in cell-attached patches 72 +/- 17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K+ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca2+]i. Both phases of increasing channel activity required the presence of extracellular Ca2+ suggesting that [Ca2+]i was mainly increased by Ca2+ influx. The N-methyl-D-aspartate (NMDA) antagonists dizocilpine (MK-801, 10 microM) and DL-2-amino-5-phosphonovaleric acid (AP5; 100 microM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca2+ through NMDA receptor channels causes a strong activation of Ca(2+)-dependent K+ channels, which is likely to result in pronounced loss of intracellular K+. NMDA receptor channels seem to remain active for a long time (> 10 min) after the end of glutamate application.
Backus
K H
KH
Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Trube
G
G
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018118:Chloride Channels / Q000187:drug effects; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004594:Electrophysiology; D005110:Extracellular Space / Q000378:metabolism; D005971:Glutamates / Q000506:poisoning*; D018698:Glutamic Acid; D007473:Ion Channels / Q000187:drug effects* / Q000502:physiology*; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology; D051381:Rats
D018118:Chloride Channels; D005971:Glutamates; D007473:Ion Channels; D015221:Potassium Channels; D018698:Glutamic Acid; D016291:Dizocilpine Maleate; D015763:2-Amino-5-phosphonovalerate; D001920:Bradykinin; D002118:Calcium