Properties of the Ryanodine‐sensitive Release Channels that Underlie Caffeine‐induced Ca²⁺ Mobilization from Intracellular Stores in Mammalian Sympathetic Neurons

Abstract

The most compelling evidence for a functional role of caffeine-sensitive intracellular Ca2+ reservoirs in nerve cells derives from experiments on peripheral neurons. However, the properties of their ryanodine receptor calcium release channels have not been studied. This work combines single-cell fura-2 microfluorometry, [3H]ryanodine binding and recording of Ca2+ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca2+ measurements showed that these cells possess caffeine-sensitive intracellular Ca2+ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage-gated calcium channels. The efficiency of caffeine in releasing Ca2+ showed a complex dependence on [Ca2+]i. Transient elevations of [Ca2+]i by 50-500 nM were facilitatory, but they became less facilitatory or depressing when [Ca2+]i reached higher levels. The caffeine-induced Ca2+ release and its dependence on [Ca2+]i was further examined by [3H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high-affinity binding site for ryanodine with a dissociation constant (KD = 10 nM) similar to that previously reported for brain microsomes. However, the density of [3H]ryanodine binding sites (Bmax = 2.06 pmol/mg protein) was at least three-fold larger than the highest reported for brain tissue. [3H]Ryanodine binding showed a sigmoidal dependence on [Ca2+] in the range 0.1-10 microM that was further increased by caffeine. Caffeine-dependent enhancement of [3H]ryanodine binding increased and then decreased as [Ca2+] rose, with an optimum at [Ca2+] between 100 and 500 nM and a 50% decrease between 1 and 10 microM. At 100 microM [Ca2+], caffeine and ATP enhanced [3H]ryanodine binding by 35 and 170% respectively, while binding was reduced by > 90% with ruthenium red and MgCl2. High-conductance (240 pS) Ca2+ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca2+ from the cytosolic side, and were blocked by Mg2+ and ruthenium red. Ryanodine (2 microM) slowed channel gating and elicited a long-lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to the subconductance level. These results show that the properties of the ryanodine receptor/Ca2+ release channels present in mammalian peripheral neurons can account for the properties of caffeine-induced Ca2+ release. Our data also suggest that the release of Ca2+ by caffeine has a bell-shaped dependence on Ca2+ in the physiological range of cytoplasmic [Ca2+].

All-or-None Ca2+Release from Intracellular Stores Triggered by Ca2+Influx through Voltage-Gated Ca2+Channels in Rat Sensory Neurons

“…This paradigm also kept the caffeine concentration, and thus the sensitivity of ryanodine receptors to Ca 2ϩ , constant. The CICR responses described here were smaller than those observed in sympathetic neurons (Hernandez-Cruz et al, 1997), in which peak [C a 2ϩ ] i values of 500 -1000 nM might produce complex kinetics for activation attributable to the binding of Ca 2ϩ to a low-affinity inactivation site on the ryanodine receptor (Bezprozvanny et al, 1991;Hernandez-Cruz et al, 1995). In the presence of caffeine, the threshold was poised to discriminate bursts of action potentials from weaker signals.…”

“…The increase in [C a 2ϩ ] i was plotted as a f unction of electrical charge transferred by C a 2ϩ (͐I C a dt). In the absence of caffeine, [C a 2ϩ ] i rose in proportion to Ca 2ϩ influx ( release channels (Bezprozvanny et al, 1991) and a strong Ca 2ϩ -dependent facilitation of ryanodine binding to the receptor in sympathetic neurons (Hernandez-Cruz et al, 1995). The discontinuity in the relationship between C a 2ϩ influx and changes in [Ca 2ϩ ] i suggested that the switch to the f ull-size response resulted from recruitment of CICR from intracellular stores.…”

Effects of SEPYLRFamide on acetylcholine-induced currents of Helix aspersa neurones: role of ryanodine receptors

“…Ruthenium red (low lM) has been reported to be an antagonist of RYRs, eectively blocking the caeineinduced elevation of intracellular Ca 2+ (Smith et al 1986;Meissner and Henderson 1987;Bezprozvanny et al 1991;Berridge 1993b;McGarry and Williams 1994;Hernandez-Cruz et al 1995;Kano et al 1995;Sitsapesan et al 1995). Ruthenium red (1 mM intracellular injection) reduced the inhibitory eect of SEPYLRFamide on the ACh-current.…”

“…Ryanodine binds preferentially to the open conformation of RYRs (altering channel-gating and locking them in an open but low conductance state) or prevents subsequent channel opening (Irving et al 1992;Bezprozvanny et al 1991;Oyamada et al 1993;Kano et al 1995;Simpson et al 1995;Sitsapesan et al 1995). Ligand-gated Ca 2+ release channels/RYRs are activated by Ca 2+ , adenine nucleotides, cyclic adenosine diphosphate ribose (cADPR), adenosine diphosphate ribose (ADPR), b-nicotinamide adenine dinucleotide (bNAD + ) and caeine; they are inhibited by Mg 2+ , H + , calmodulin and ruthenium red (Smith et al 1986;Meissner 1986;Meissner and Henderson 1987;Bezprozvanny et al 1991;Irving et al 1992;Oyamada et al 1993;McGarry and Williams 1994;Hernandez-Cruz et al 1995;Simpson et al 1995;Sitsapesan et al 1995). Several distinct sites of ligand action occur on RYRs (McGarry and Williams 1994;Simpson et al 1994;Sitsapesan et al 1995).…”

Structure-function relations in dendritic spines: Is size important?

“…Functionally, stores can be caused to release bound calcium into the intracellular space by two distinct receptors, the ryanodine receptor and the IP-3 receptor. The former has been found in dendritic spines of cultured hippocampal neurons, and is known to be activated directly by caffeine (Hernandez-Cruz et al, 1995). Caffeine-evoked calcium transients have been described in the hippocampus (Garaschuk et al, 1997), and the machinery for caffeine-induced calcium release from dendritic spines has been studied by Korkotian and Segal (1998).…”