CHANGES IN [CA2-NEURONS(](I) AND MEMBRANE CURRENTS DURING IMPAIRED MITOCHONDRIAL METABOLISM IN DISSOCIATED RAT HIPPOCAMPAL)

1. This study was designed to establish the basis for altered membrane excitability during the inhibition of mitochondrial metabolism in cen tral mammalian neurons. Perforated whole-cell patch clamp and fluorime tric techniques were combined to examine changes in membrane currents, intracellular calcium ([Ca2+](i)) and mitochondrial potential (Delta Psi(m)) in neurons dissociated from the CA1 subfield of the hippocampu s 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 reve rsal potential of the current at -83 mV and its dependence on extracel lular [K+] confirmed that this was a K+ conductance. 3. Simultaneous r ecordings of [Ca2+](i) and current showed a striking correlation betwe en a rise in [Ca2+](i) and the developed outward current. Flash photol ysis of the caged Ca2+ chelator, diazo-2, reversed both the rise in [C a2+](i) and the outward current. We current was reduced by 80% by char ybdotoxin, was attenuated by 10 mM TEA(+) but was unaffected by apamin or by the K-ATP channel blocker tolbutamide (400 mu M-1 mM). These da ta suggest strongly that the current is carried by Ca2+-dependent K+ c hannels. 4. Simultaneous recordings of membrane current, Delta Psi(m) and [Ca2+](i) revealed the sequence of events in response to impaired mitochondrial function (CN, FCCP or anoxia): Delta Psi(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 m itochondrial repolarization. 5. The rise in [Ca2+](i) appeared to resu lt from an increased Ca2+ influx through voltage-gated Ca2+ channels. It was dependent on extracellular Ca2+ and was much reduced by methoxy verapamil (D600). The rate of Mn2+ quench of fura-2 fluorescence was i ncreased 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 o n membrane potential in cells clamped at a range of holding potentials from -90 to -45 mV. 6. Thus, removal of oxygen, uncoupling mitochondr ial oxidative phosphorylation or inhibition of respiration, all lead t o mitochondrial depolarization, an increased Ca2+ influx through (volt age-gated) channels, even at hyperpolarized membrane potentials, raisi ng [Ca2+](i) which in turn drives an increased K+ conductance that mod ulates membrane excitability.