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

D.R. Laver1, G.D. Lamb2, 1Biomedical Sciences, University of Newcastle, Callaghan, Australia, 2Zoology, LaTrobe University, Melbourne, VIC, Australia

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.