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Changes in [Ca²⁺]_i and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons

Alex V. Nowicky * and Michael R. Duchen

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 (_m) 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 K_ATP channel blocker tolbutamide (400 µM-1 mM). These data suggest strongly that the current is carried by Ca²⁺-dependent K⁺ channels.

4. Simultaneous recordings of membrane current, _m and [Ca²⁺]_i revealed the sequence of events in response to impaired mitochondrial function (CN, FCCP or anoxia): _m 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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