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Simultaneous whole-cell patch-clamp and Fura-2 microfluorimetric recordings of calcium currents (ICa) and the intracellular free Ca2+ concentration (Ca2+i) were made from neurones grown in primary culture from the dorsal root ganglion of the rat. 2. Cells held at -80 mV and depolarized to 0 mV elicited a ICa that resulted in an Ca2+i transient which was not significantly buffered during the voltage step and lasted long after the cell had repolarized and the current ceased. The process by which the cell buffered Ca2+i back to basal levels could best be described with a single-exponential equation. 3. The membrane potential versus ICa and Ca2+i relationship revealed that the peak of the Ca2+i transient evoked at a given test potential closely paralleled the magnitude of the ICa suggesting that neither voltage-dependent nor Ca2(+)-induced Ca2+ release from intracellular stores made a significant contribution to the Ca2+i transient. 4. When the cell was challenged with Ca2+ loads of different magnitude by varying the duration or potential of the test pulse, Ca2+i buffering was more effective for larger Ca2+ loads. The relationship between the integrated ICa and the peak of the Ca2+i transient reached an asymptote at large Ca2+ loads indicating that Ca2(+)-dependent processes became more efficient or that low-affinity processes had been recruited. 5. Inhibition of Ca2+ influx with neuropeptide Y demonstrated that inhibition of a large ICa produced minor alterations in the peak of the Ca2+i transient, while inhibition of smaller currents produced corresponding decreases in the Ca2+i transient. Thus, inhibition of the ICa was reflected by a change in the peak Ca2+i only when submaximal Ca2+ loads were applied to the cell, implying that modulation of Ca2+i is dependent on the activation state of the cells. 6. Intracellular dialysis with the mitochondrial Ca2+ uptake blocker Ruthenium Red in whole-cell patch-clamp experiments removed the buffering component which was responsible for the more efficient removal of Ca2+i observed when large Ca2+ loads were applied to the cell. 7. When cells were superfused with 50 mM-K+, Ca2+i transients recorded from the cell soma returned to control levels very slowly. Pharmacological studies indicated that mitochondria were cycling Ca2+ during this sustained elevation in Ca2+i. In contrast, Ca2+i transients recorded from cell processes returned to basal levels relatively rapidly. 8. Extracellular Na(+)-dependent Ca2+ efflux did not significantly contribute to buffering Ca2+i transients in dorsal root ganglion neurone cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)
Thayer et al. (Fri,) studied this question.