Programme Contents

Regulation of ryanodine receptors from cardiac muscle by luminal Ca²⁺ and Mg²⁺

Muscle contraction occurs when Ca²⁺ is released from the sarcoplasmic reticulum (SR) through ryanodine receptor Ca²⁺ release channels (RyRs). In heart, uptake and release of Ca²⁺ from the SR causes the free [Ca²⁺] within the lumen ([Ca²⁺]_L) to cycle between ∼0.3 to 1.0 mmol/l during the normal heart beat (Ginsburg et al., 1998). [Ca²⁺]_L is known to regulate the Ca²⁺ releasing excitability of this store by stimulating the RyRs in its membrane. The resulting negative feedback between store depletion and Ca²⁺ release is believed to drive pacemaking and rythmicity cardiac muscle (Vinogradova et al., 2005) as well as smooth muscle (Van Helden, 1993) and neurons (Verkhratsky, 2005). Luminal stimulation of RyRs involves three Ca²⁺ sensing mechanisms on both the luminal and cytoplasmic side of the RyR (Laver, 2007); namely the luminal Ca²⁺-activation site (L-site, 60 μmol/l affinity), the cytoplasmic activation site (A-site, 0.9 μmol/l affinity) and the high affinity cytoplasmic Ca²⁺-inactivation site (I₂-site, 1.2 μmol/l affinity). Cardiac RyR (RyR2 isoform) activation by luminal Ca²⁺ occurs by a multi-step process dubbed "luminal-triggered Ca²⁺ feed-through". Ca²⁺ binding to the L-site initiates channel openings where upon luminal Ca²⁺ can flow through to the A-site (producing prolongation of openings) and to the I₂-site (causing inactivation at high levels of Ca²⁺ feed-through). Cytoplasmic Mg²⁺ inhibits RyRs by displacing Ca²⁺ from the A-site (Laver et al., 1997) and plays an important role in regulating Ca²⁺ release. However, the possibility that similar processes occur at the L- and I₂-sites has not been explored.
To explore this possibility, single RyRs and RyR arrays were incorporated into artificial lipid bilayers. SR vesicles were prepared from sheep hearts. Animals were killed by barbiturate overdose prior to muscle removal. SR vesicles containing RyRs were incorporated into artificial planar lipid bilayers which separated baths corresponding to the cytoplasm and SR lumen. The baths contained 230 mmol/l CsCH₃O₃S, 20 mmol/l CsCl, 10 mmol/l TES (pH 7.4) plus various amounts of Ca²⁺, Mg²⁺ and ATP. Channel activity was recorded using Cs⁺ as the current carrier. A novel, high affinity inhibition of RyR2 by luminal Mg²⁺ was observed, pointing to an important physiological role for luminal Mg²⁺ in cardiac muscle. At diastolic cytoplasmic [Ca²⁺] ([Ca²⁺]_C = 100 nmol/l) luminal Mg²⁺ inhibition was voltage-independent and was alleviated by increasing luminal [Ca²⁺]. The K_i for Mg²⁺ inhibition increased from 90 μmol/l at [Ca²⁺]_L = 0.3 mmol/l to 1 mmol/l at [Ca²⁺]_L = 1 mmol/l. At systolic [Ca²⁺]_C (1- 10 μmol/l), Mg²⁺ inhibition was substantially reduced and its properties were consistent with luminal Mg²⁺ flowing through the channel and binding to the cytoplasmic A-site. Under these conditions K_i was voltage-dependent; 13 mmol/l at -40 mV and >100 mmol/l at +40 mV. The data could be accurately fitted by a model in which Mg²⁺ and Ca²⁺ compete at both the L- and A-sites and where the L-site has similar affinities for both ions. The model predicts that under physiological divalent ion concentrations (1 mmol/l free Mg²⁺ in the cytoplasm and lumen) and membrane potential (0 mV), [Ca²⁺]_L activation of Ca²⁺ release is primarily due to displacement of Mg²⁺ from the L-site and that luminal Mg²⁺ is an essential cofactor for the phenomenon. Therefore competition between luminal Ca²⁺ and Mg²⁺ may play an essential role in store-load dependent Ca²⁺ release.

Ginsburg KS, Weber CR & Bers DM. (1998) Journal General Physiology, 111: 491-504.

Vinogradova TM, Maltsev VA, Bogdanov KY, Lyashkov AE & Lakatta EG. (2005) Annals of the New York Academy of Sciences, 1047: 138-156

Van Helden DFJ. (1993) Journal of Physiology, 471: 465-79.

Verkhratsky A.(2005) Physiological Reviews, 85: 201-79.

Laver DR. (2007) Biophysical Journal, 92: 3541-55.

Laver DR, Baynes TM & Dulhunty AF. Journal of Membrane Biology, 156: 213-29.