Programme

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

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.