| 9490829 |
Nowicky AV, Duchen MR: Changes in [Ca2+] i and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons. J Physiol. 1998 Feb 15;507 ( Pt 1):131-45.
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. |
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