title: Changes in [Ca2+]i and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons.
abstract: 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.
authors.affiliation: School of Health, Biology and Environmental Sciences, Middlesex University, Queensway, Enfield, London, EN3 4SF, UK.
authors.forename: A V
authors.initials: AV
authors.lastname: Nowicky
authors.forename: M R
authors.initials: MR
authors.lastname: Duchen
mesh_terms: 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
chemical_list: D004791:Enzyme Inhibitors; D015221:Potassium Channels; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D008345:Manganese; D012966:Sodium Cyanide; D011188:Potassium; D002118:Calcium