Single Ryanodine Receptor Channel Basis of Caffeine's Action on Ca2+ Sparks

Abstract

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.

K201 (JTV519) is a Ca2+-Dependent Blocker of SERCA and a Partial Agonist of Ryanodine Receptors in Striated Muscle

“…We also tested the effect of K201 on channels incubated with cytosolic [Ca 21 ] ∼100 nM in the presence of Mg 21 /ATP 5 mM. As previously reported Porta et al, 2011), RyR2 display sporadic openings and are quiescent most of the time under these conditions (P o , 0.01). Aliquot additions of K201 (from 1.5 to 80 mM) did not alter RyR2 gating kinetics or channel activity Fig.…”

Eudistomin D and Penaresin Derivatives as Modulators of Ryanodine Receptor Channels and Sarcoplasmic Reticulum Ca2+ATPase in Striated Muscle

“…Of the many pharmacologic agents that modulate RyRs (Conley and Brammar, 1996;West and Williams, 2007), the diagnostic ligand caffeine particularly raised our interest due to its ability to target domains that stabilize RyRs in high-P o mode (Rousseau and Meissner, 1989;Sitsapesan and Williams, 1990;Porta et al, 2011). However, caffeine and other xanthines are inadequate for binding studies or molecular labeling due to their relatively low affinity for RyRs (Liu and Meissner, 1997).…”

Microfluidic platform for bilayer experimentation

“…Membranes prepared by the painting technique, 1 which are defined as solvent containing membranes, are characterized by Cs ≈ 0.45 µF/cm 2 against 0.9 µF/cm 2 and 1.0 µF/cm 2 for solvent-less and cell membranes, respectively. 39 The value found here suggests that the membranes formed in our microfluidic device may contain less solvent than those prepared with the painting technique. Following this, the thickness of the membrane is derived using d=ε0εr/Cs where εr is the dielectric constant (relative permittivity) of the bilayer and ε0 the permittivity of free space.…”

“…For instance, the ryanodine receptor (RyR) is an intracellular calcium channel involved in cardiovascular diseases, which is located in the membrane of the sarcoplasmic reticulum (ER). 38 This ion channel has been studied in a bilayer format for its electrophysiological characterization, 39 which has brought better insight into its function, through single RyR channel recordings. Measurements conducted on cells using a fluorescence approach only provide collective data on a channel population, with possible influence of RyR protein channels on each other.…”

“…Measurements conducted on cells using a fluorescence approach only provide collective data on a channel population, with possible influence of RyR protein channels on each other. 39 However, a limiting step found in the bilayer approach is the reconstitution of the ion channels in the bilayer matrix. For that essential step, proteins are most of the time first expressed in a cell system, for which still a dedicated cell culture facility is required.…”