Single cardiac sarcoplasmic reticulum Ca2+-release channel: activation by caffeine

Abstract

Caffeine is thought to affect excitation-contraction coupling in cardiac muscle by activating the sarcoplasmic reticulum (SR) Ca2+-release channel. The effect of caffeine at the single channel level was studied by incorporating canine cardiac SR vesicles into planar lipid bilayers. Cardiac Ca2+-release channels were activated in a steady-state manner by millimolar cis-caffeine and displayed a unitary conductance (77 pS in 50 mM Ca2+ trans) similar to that previously observed for the Ca2+-activated cardiac channel. The caffeine-activated channel was moderately sensitive to the voltage applied across the bilayer, was sensitive to further activation by ATP, and was inhibited by Mg2+ and ruthenium red. Kinetic analysis showed that at low Ca2+ concentration, caffeine activated the channel by increasing the frequency and the duration of open events.

Evidence for novel caffeine and Ca2+ binding sites on the lobster skeletal ryanodine receptor

“…; n=5). This value is similar to those previously reported for mammalian isoforms of RyR (Smith et al, 1986;Rousseau & Meissner, 1989;Sitsapesan & Williams, 1990) under the same ionic conditions. Our preliminary data therefore suggests that the ion handling properties of the lobster RyR may be very similar to those observed in other RyR isoforms.…”

“…Thus the basic gating scheme for the Ca 2+ -activated lobster skeletal RyR deviates signi®cantly from that reported for mammalian skeletal isoforms of RyR. The mechanism for Ca 2+ activation appears to be more similar to that reported for dog cardiac RyR (Rousseau & Meissner, 1989;Chu et al, 1993) where the open events are generally longer and Ca 2+ as the sole activator causes large increases in the open lifetime duration in addition to an increase in the frequency of channel opening.…”

“…Ca eine, at concentrations (10 ± 40 mM) that will virtually fully open mammalian isoforms of RyR (Rousseau et al, 1988;Rousseau & Meissner, 1989;Sitsapesan & Williams, 1990;Chen et al, 1997) in the presence of activating cytosolic [Ca 2+ ], did not cause very large changes in the Po of lobster skeletal RyR (see Figures 7 and 8). It has been suggested that low concentrations (0.5 ± 2 mM) of agents acting via the ca eine binding sites on sheep cardiac RyR sensitize the channel to Ca 2+ (Sitsapesan & Williams, 1990;Williams & Holmberg, 1990) but that high concentrations of ca eine or related compounds can, in the presence of activating cytosolic [Ca 2+ ], fully activate the channel by invoking a di erent gating scheme (Sitsapesan & Williams, 1990;Williams & Holmberg, 1990;McGarry & Williams, 1994).…”

“…While ca eine can signi®cantly increase Po in the presence of 10 mM Ca 2+ , ca eine (10 mM) does not even increase Po to the maximal level observed with Ca 2+ as the sole channel activator (see Figure 4) and at 700 mM Ca 2+ , ca eine does not cause any further increases in Po. Thus the e ect of ca eine on the gating of the lobster RyR is very di erent to its e ect on the gating of mammalian isoforms of RyR where the channels can be fully opened by ca eine at activating [Ca 2+ ] and can be activated to a lower level at sub-activating [Ca 2+ ] (Rousseau et al, 1988;Rousseau & Meissner, 1989;Sitsapesan & Williams, 1990).…”

Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation

“…To block the SR-dependent component of relaxation, it is possible to evoke Ca 2+ transients by rapid application of caffeine to quiescent cells. Because caffeine at millimolar concentrations promotes SR Ca 2+ channel opening (6), not only does it release Ca 2+ from the SR, but also prevents significant Ca 2+ accumulation in the organelle, which is equivalent to inhibition of the SR-A. In this case, relaxation would rely only on NCX and the so-called slow systems (i.e., sarcolemmal Ca 2+ -ATPase and the mitochondrial Ca 2+ uniporter).…”