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Regulation of coupled calcium release channels in bilayers by luminal Ca²⁺

Contraction in skeletal and cardiac muscle occurs when Ca²⁺ is released from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) Ca²⁺ release channels. In muscle, the RyRs are activated by ATP in the cytoplasm and by Ca²⁺ in the cytoplasm and lumen of the SR. This study investigates their mechanisms of action.

RyRs were isolated from rabbit skeletal muscle that had been removed from dead rabbits. RyRs were incorporated into artificial planar lipid bilayers separating baths corresponding to the cytoplasm and SR lumen. Single channel activity was recorded using Cs⁺ as the current carrier.

Cytoplasmic Mg²⁺ is a potent inhibitor of RyRs. Although it is recognised that Mg²⁺ binds at the cytoplasmic Ca²⁺ sites, it is not clear if Mg²⁺ is an antagonist or merely prevents Ca²⁺ from activating the channel. This distinction becomes important considering that physiological [ATP] activates skeletal RyRs in the absence of cytoplasmic Ca²⁺. We measured the effects of Mg²⁺-inhibition on ATP activated RyRs in the absence of Ca²⁺ and found that Mg²⁺ is indeed a RyR antagonist.

RyRs showed coupled gating when conditions favoured Ca²⁺ flow from the luminal to cytoplasmic baths (i.e. the rate constant for channel opening was increased by the opening of neighbouring RyRs in the bilayer). This indicates that RyRs can be in close proximity and that luminal Ca²⁺ can permeate open channels to activate neighbouring RyRs at the cytoplasmic activation sites. Curiously, we find that Ca²⁺ released by a RyR has a greater stimulatory effect on its neighbours than itself.

We describe a novel mechanism for luminal Ca²⁺ regulation of Ca²⁺ release whereby increasing luminal [Ca²⁺] decreases the apparent affinity of the RyR for cytoplasmic Mg²⁺. This decrease in apparent Mg²⁺ affinity was not due to competition between luminal Ca²⁺ and cytoplasmic Mg²⁺. Rather, it appears that luminal Ca²⁺ can regulate RyRs via an allosteric mechanism independently of Ca²⁺ flow through the channel.