Effect of R56865 on cardiac sarcoplasmic reticulum function and its role as an antagonist of digoxin at the sarcoplasmic reticulum calcium release channel
Authors: McGarry Stephen J. , Scheufler Eckhard , Williams Alan J. 
Published: July 2012

The effect of R56865 (N‐[1‐[4‐(4‐fluorophenoxy)‐butyl]‐4‐piperidinyl]‐N‐methyl‐2‐benzothiazolamine) on cardiac sarcoplasmic reticulum (SR) Ca2+‐release channel function was investigated. The effect of R56865 on [3H]‐ryanodine and [3H]‐digoxin binding to SR vesicles and its effect on the ATP‐stimulated 45Ca2+ uptake into SR vesicles was also studied.

R56865 (0.5–50 μm) had no effect on single‐channel open probability (Po) when added to native cardiac SR Ca2+‐release channels, incorporated into planar phospholipid bilayers, that had previously been activated by 10 μm Ca2+. The single‐channel conductance (93 pS) and the Ca2+/Tris+ permeability ratio (12.5) were also unaffected by R56865.

R56865 failed to affect the rapid Ca2+‐induced efflux of 45Ca2+ from cardiac SR vesicles. The initial efflux rate at an extravesicular [Ca2+] of 0.1 μm was 176 ± 33 nmol 45Ca2+ mg−1 proteins s1 (n = 5). Addition of 0.5–50 μm R56865 to the efflux solution did not affect the initial efflux rate or the total amount of 45Ca2+ released from the vesicles.

The specific binding of [3H]‐ryanodine to SR vesicles can be viewed as a marker for SR Ca2+‐release channel activation. R56865 (0.05–50 μm) did not change the amount of specific [3H]‐ryanodine bound at 10 μm activating Ca2+. Taken together these data (points 2, 3 and 4) suggest that R56865 does not affect the Ca2+ activation of the cardiac SR Ca2+‐release channel.

R56865 (0.5–50 μm) decreased the ATP‐stimulated uptake of 45Ca2+ into cardiac SR vesicles. The total amount of 45Ca2+ taken up into the vesicles was decreased by 26–37% and the initial rate of uptake was also reduced.

R56865 decreased the binding of [3H]‐digoxin to cardiac SR vesicles. It inhibited binding of [3H]‐digoxin to both sites on SR membranes. However, it acted as a mixed type of non‐competitive antagonist, as it increased the Kd and reduced the Bmax for [3H]‐digoxin. Additionally, when SR Ca2+‐release channels incorporated into bilayers were activated by 1 nm digoxin at 10 μm Ca2+, 5 μm R56865 decreased single‐channel Po to that seen prior to activation by digoxin, confirming that R56865 is an antagonist at the cardiac glycoside receptor site on the cardiac SR Ca2+‐release channel.

The results presented here show that R56865 decreases 45Ca2+ uptake into SR vesicles and also non‐competitively decreases the binding of cardiac glycosides to their activation sites on the SR Ca2+‐release channel. Since this compound fails to affect other aspects of the functioning of the SR, we conclude that inhibition of Ca2+‐uptake into the SR and/or antagonism of digoxin binding to its high affinity sites on the cardiac SR Ca2+‐release channel may contribute to the protection against glycoside‐induced toxicity seen with R56865. Indeed the reduction of Ca2+ uptake into the SR during Ca2+ overload may contribute to the protective action of R56865 against arrhythmias induced by veratridine or by ischaemia. Notwithstanding this, it is possible that the multitude of other actions on specific ion translocation processes, as well as the possible non‐specific membrane effects of R56865, may be as responsible for the protection against arrhythmias as the effects on SR function we have documented here. However, the simultaneous interference with more than one of these processes by R56865 may be required for its anti‐arrhythmic action.