TY - JOUR
T1 - Release and sequestration of calcium by ryanodine-sensitive stores in rat hippocampal neurones
AU - Garaschuk, Olga
AU - Yaari, Yoel
AU - Konnerth, Arthur
PY - 1997/7/1
Y1 - 1997/7/1
N2 - 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 Ca2+-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 Ca2+-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+], returns to its resting level. 8. Pressure applications of caffeine onto pyramidal cell dendrites evoked local C2+ 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 C2+ stores in hippocampal pyramidal neurones contain a releasable pool of Ca2+ that is maintained by a Ca2+ entry pathway active at subthreshold membrane potentials. C2+ 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.
AB - 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 Ca2+-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 Ca2+-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+], returns to its resting level. 8. Pressure applications of caffeine onto pyramidal cell dendrites evoked local C2+ 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 C2+ stores in hippocampal pyramidal neurones contain a releasable pool of Ca2+ that is maintained by a Ca2+ entry pathway active at subthreshold membrane potentials. C2+ 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.
UR - http://www.scopus.com/inward/record.url?scp=0030825675&partnerID=8YFLogxK
U2 - 10.1111/j.1469-7793.1997.013bl.x
DO - 10.1111/j.1469-7793.1997.013bl.x
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
C2 - 9234194
AN - SCOPUS:0030825675
SN - 0022-3751
VL - 502
SP - 13
EP - 30
JO - Journal of Physiology
JF - Journal of Physiology
IS - 1
ER -