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The response of the ryanodine receptor to reduced luminal Ca2+ concentrations is depressed by calsequestrin

L. Wei1, D.R. Laver2, N.A. Beard1, A.F. Dulhunty1, 1Division of Molecular Biosciences, John Curtin School of Medical Research, ANU, Canberra, ACT, Australia, 2Faculty of Health, School of Biomedical Science, University of Newcastle, Newcastle, NSW, Australia

The ryanodine receptor (RyR) Ca2+ release channel in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle is essential for excitation-contraction (EC) coupling. Calsequestrin (CSQ) is the major Ca2+ binding protein in the SR and also regulates RyRs. Since Ca2+ release from SR is determined by the Ca2+ load, CSQ is possibly a luminal Ca2+ sensor for the RyR. There are contradictory reports about the effect of changing luminal free Ca2+ concentration ([Ca2+]free) on RyR activity, which are unexplained but may depend on the channels association with CSQ.

To investigate the responses of RyRs to altering luminal [Ca2+]free in the presence and absence of CSQ, rabbit skeletal SR vesicles (from freshly euthanased rabbits) were reconstituted in artificial lipid bilayers, which separates two chambers, denoted cytoplasmic and luminal respectively. Luminal [Ca2+]free was adjusted between 1 mM to 100 nM by adding BAPTA or EGTA, and channel activities were tested in both CSQ-associated and CSQ-dissociated RyRs at both sub-activating (100 nM) and activating (50 m M) cytoplasmic Ca2+.

Lowering luminal [Ca2+]free from 1 mM to 100 nM resulted in immediate activation of RyRs in CSQ-dissociated RyRs. In contrast, either less increase or in fact decreased activity was observed in CSQ-associated RyRs when luminal Ca2+ was decreased. The changes were independent of initial channel activity and the type of Ca2+ chelator.

The data show that the RyR response to changing luminal [Ca2+]free depends on CSQ association. The activation by a fall in luminal [Ca2+]free was depressed in the presence of CSQ. This suggests that CSQ acts as a luminal Ca2+ sensor for the RyR at lower than physiological [Ca2+]s and could effectively reduce excess Ca2+ release from the SR during stress or fatigue and thus act to conserve the store load.