Release and sequestration of calcium by ryanodine-sensitive stores in rat hippocampal neurones.
1. The properties of ryanodine-sensitive Ca2+ stores in CA1 pyramidal cells were investigated in rat hippocampal slices by using whole-cell patch-clamp recordings combined with fura-2-based fluorometric digital imaging of cytoplasmic Ca2+ concentration ([Ca2+]i). 2. Brief pressure applications of caffeine onto the somata of pyramidal cells caused large transient increases in [Ca2+]i (Ca2+ transients) of 50-600 nM above baseline. 3. The Ca2+ transients evoked by caffeine at -60 mV were not associated with an inward current, persisted after blocking voltage-activated Ca2+ currents and were completely blocked by bath-applied ryanodine. Similar transients were also evoked at +60 mV. Thus, these transients reflect Ca2+ release from intracellular ryanodine-sensitive Ca2+ stores. 4. The Ca2+ transients evoked by closely spaced caffeine pulses rapidly decreased in amplitude, indicating progressive depletion of the Ca2+ stores. The amplitude of the Ca2+ transients recovered spontaneously with an exponential time constant of 59 s. Recovery was accelerated by depolarization-induced elevations in [Ca2+]i and blocked by cyclopiazonic acid (CPA) and thapsigargin, indicating that store refilling is mediated by endoplasmic reticulum Ca(2+)-ATPases. 5. Even without prior store depletion the caffeine-induced Ca2+ transients disappeared after 6 min exposure to CPA, suggesting that ryanodine-sensitive Ca2+ stores are maintained at rest by continuous Ca2+ sequestration. 6. Caffeine-depleted Ca2+ stores did not refill in Ca(2+)-free saline, suggesting that the refilling of the stores depends upon Ca2+ influx through a 'capacitative-like' transmembrane influx pathway operating at resting membrane potential. The refilling of the stores was also blocked by Ni2+ and gallopamil (D600). 7. Elevations of basal [Ca2+]i produced by bath-applied KCl markedly potentiated (up to 6-fold) the caffeine-induced Ca2+ transients. The degree of potentiation was positively related to the increase in basal [Ca2+]i. The Ca2+ transients remained potentiated up to 9 min after reversing the KCl-induced [Ca2+]i increase. Thus, the ryanodine-sensitive Ca2+ stores can 'overcharge' when challenged with an increase in [Ca2+]i and slowly discharge excess Ca2+ after basal [Ca2+]i returns to its resting level. 8. Pressure applications of caffeine onto pyramidal cell dendrites evoked local Ca2+ transients similar to those separately evoked in the respective somata. Thus, dendritic ryanodine-sensitive Ca2+ stores are also loaded at rest and can function as independent compartments. 9. In conclusion, the ryanodine-sensitive Ca2+ stores in hippocampal pyramidal neurones contain a releasable pool of Ca2+ that is maintained by a Ca2+ entry pathway active at subthreshold membrane potentials. Ca2+ entry through voltage-gated Ca2+ channels transiently overcharges the stores. Thus, by acting as powerful buffers at rest and as regulated sources during activity, Ca2+ stores may control the waveform of physiological Ca2+ signals in CA1 hippocampal pyramidal neurones.
Garaschuk
O
O
I Physiologisches Institut, Universität des Saarlandes, Homburg, Germany.
Yaari
Y
Y
Konnerth
A
A
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D003712:Dendrites / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology* / Q000502:physiology; D007091:Image Processing, Computer-Assisted; D007211:Indoles / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009124:Muscle Proteins / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D017966:Pyramidal Cells / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D019837:Ryanodine Receptor Calcium Release Channel; D013569:Synapses / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D019284:Thapsigargin / Q000494:pharmacology
D015220:Calcium Channels; D002148:Calmodulin-Binding Proteins; D000697:Central Nervous System Stimulants; D004791:Enzyme Inhibitors; D007211:Indoles; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D019284:Thapsigargin; D011188:Potassium; D002118:Calcium; C000543:cyclopiazonic acid