Release and sequestration of calcium by ryanodine‐sensitive stores in rat hippocampal neurones

Abstract

The properties of ryanodine‐sensitive Ca 2+ 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 Ca 2+ concentration ([Ca 2+]i). Brief pressure applications of caffeine onto the somata of pyramidal cells caused large transient increases in [Ca 2+]i (Ca 2+ transients) of 50–600 n m above baseline. The Ca 2+ transients evoked by caffeine at −60 mV were not associated with an inward current, persisted after blocking voltage‐activated Ca 2+ currents and were completely blocked by bath‐applied ryanodine. Similar transients were also evoked at +60 mV. Thus, these transients reflect Ca 2+ release from intracellular ryanodine‐sensitive Ca 2+ stores. The Ca 2+ transients evoked by closely spaced caffeine pulses rapidly decreased in amplitude, indicating progressive depletion of the Ca 2+ stores. The amplitude of the Ca 2+ transients recovered spontaneously with an exponential time constant of 59 s. Recovery was accelerated by depolarization‐induced elevations in [Ca 2+]i and blocked by cyclopiazonic acid (CPA) and thapsigargin, indicating that store refilling is mediated by endoplasmic reticulum Ca 2+‐ATPases. Even without prior store depletion the caffeine‐induced Ca 2+ transients disappeared after 6 min exposure to CPA, suggesting that ryanodine‐sensitive Ca 2+ stores are maintained at rest by continuous Ca 2+ sequestration. Caffeine‐depleted Ca 2+ stores did not refill in Ca 2+‐free saline, suggesting that the refilling of the stores depends upon Ca 2+ influx through a ‘capacitative‐like’ transmembrane influx pathway operating at resting membrane potential. The refilling of the stores was also blocked by Ni 2+ and gallopamil (D600). Elevations of basal [Ca 2+]i produced by bath‐applied KCl markedly potentiated (up to 6‐fold) the caffeine‐induced Ca 2+ transients. The degree of potentiation was positively related to the increase in basal [Ca 2+]i. The Ca 2+ transients remained potentiated up to 9 min after reversing the KCl‐induced [Ca 2+]i increase. Thus, the ryanodine‐sensitive Ca 2+ stores can ‘overcharge’ when challenged with an increase in [Ca 2+]i and slowly discharge excess Ca 2+ after basal [Ca 2+]i returns to its resting level. Pressure applications of caffeine onto pyramidal cell dendrites evoked local Ca 2+ transients similar to those separately evoked in the respective somata. Thus, dendritic ryanodine‐sensitive Ca 2+ stores are also loaded at rest and can function as independent compartments. In conclusion, the ryanodine‐sensitive Ca 2+ stores in hippocampal pyramidal neurones contain a releasable pool of Ca 2+ that is maintained by a Ca 2+ entry pathway active at subthreshold membrane potentials. Ca 2+ entry through voltage‐gated Ca 2+ channels transiently overcharges the stores. Thus, by acting as powerful buffers at rest and as regulated sources during activity, Ca 2+ stores may control the waveform of physiological Ca 2+ signals in CA1 hippocampal pyramidal neurones.