Changes in [Ca2+]i and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons.
1. This study was designed to establish the basis for altered membrane excitability during the inhibition of mitochondrial metabolism in central mammalian neurons. Perforated whole-cell patch clamp and fluorimetric techniques were combined to examine changes in membrane currents, intracellular calcium ([Ca2+]i) and mitochondrial potential (DeltaPsim) in neurons dissociated from the CA1 subfield of the hippocampus of young rats. 2. On application of the mitochondrial inhibitor NaCN, or the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), the membrane potential hyperpolarized and membrane conductance increased. Under voltage clamp, an outward current was seen. The reversal potential of the current at -83 mV and its dependence on extracellular [K+] confirmed that this was a K+ conductance. 3. Simultaneous recordings of [Ca2+]i and current showed a striking correlation between a rise in [Ca2+]i and the developed outward current. Flash photolysis of the caged Ca2+ chelator, diazo-2, reversed both the rise in [Ca2+]i and the outward current. The current was reduced by 80 % by charybdotoxin, was attenuated by 10 mM TEA+ but was unaffected by apamin or by the KATP channel blocker tolbutamide (400 microM-1 mM). These data suggest strongly that the current is carried by Ca2+-dependent K+ channels. 4. Simultaneous recordings of membrane current, DeltaPsim and [Ca2+]i revealed the sequence of events in response to impaired mitochondrial function (CN, FCCP or anoxia): DeltaPsim depolarized, followed rapidly by an increase in [Ca2+]i followed in turn by the outward current. [Ca2+]i and membrane current recovered only after mitochondrial repolarization. 5. The rise in [Ca2+]i appeared to result from an increased Ca2+ influx through voltage-gated Ca2+ channels. It was dependent on extracellular Ca2+ and was much reduced by methoxyverapamil (D600). The rate of Mn2+ quench of fura-2 fluorescence was increased by the inhibitors, and the inhibitors induced a small inward current when K+ channels were blocked that preceded the rise in [Ca2+]i. However, the increase in [Ca2+]i showed no obvious dependence on membrane potential in cells clamped at a range of holding potentials from -90 to -45 mV. 6. Thus, removal of oxygen, uncoupling mitochondrial oxidative phosphorylation or inhibition of respiration, all lead to mitochondrial depolarization, an increased Ca2+ influx through (voltage-gated) channels, even at hyperpolarized membrane potentials, raising [Ca2+]i which in turn drives an increased K+ conductance that modulates membrane excitability.
Nowicky
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School of Health, Biology and Environmental Sciences, Middlesex University, Queensway, Enfield, London, EN3 4SF, UK.
Duchen
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D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007425:Intracellular Membranes / Q000378:metabolism*; D008345:Manganese / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D015221:Potassium Channels / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012966:Sodium Cyanide / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D004791:Enzyme Inhibitors; D015221:Potassium Channels; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D008345:Manganese; D012966:Sodium Cyanide; D011188:Potassium; D002118:Calcium