Effects of cytoplasmic and luminal pH on Ca(2+) release channels from rabbit skeletal muscle.

Ryanodine receptor (RyR)-Ca(2+) release channels from rabbit skeletal muscle were incorporated into lipid bilayers. The effects of cytoplasmic and luminal pH were studied separately over the pH range 5-8, using half-unit intervals. RyR activity (at constant luminal pH of 7.5) was inhibited at acidic cytoplasmic pH, with a half-inhibitory pH (pH(I)) approximately 6.5, irrespective of bilayer potential and of whether the RyRs were activated by cytoplasmic Ca(2+) (50 microM), ATP (2 or 5 mM), or both. Inhibition occurred within approximately 1 s and could be fully reversed within approximately 1 s after brief inhibition or within approximately 30-60 s after longer exposure to acidic cytosolic pH. There was no evidence of any hysteresis in the cytoplasmic pH effect. Ryanodine-modified channels were less sensitive to pH inhibition, with pH(I) at approximately 5.5, but the inhibition was similarly reversible. Steady-state open and closed dwell times of RyRs during cytoplasmic pH inhibition suggest a mechanism where the binding of one proton inhibits the channel and the binding of two to three additional protons promotes further inhibited states. RyR activity was unaffected by luminal pH in the pH range 7.5 to 6.0. At lower luminal pH (5-5.5) most RyRs were completely inhibited, and raising the pH again produced partial to full recovery in only approximately 50% of cases, with the extent of recovery not detectably different between pH 7.5 and pH 9. The results indicate that isolated skeletal muscle RyRs are not inhibited as strongly by low cytoplasmic and luminal pH, as suggested by previous single-channel studies.

Cardiac ryanodine receptor activity is altered by oxidizing reagents in either the luminal or cytoplasmic solution.

The location of reactive cysteine residues on the ryanodine receptor (RyR) calcium release channel was assessed from the changes in channel activity when oxidizing or reducing reagents were added to the luminal or cytoplasmic solution. Single sheep cardiac RyRs were incorporated into lipid bilayers with 10(-7) m cytoplasmic Ca2+. The thiol specific-lipophilic-4,4'-dithiodipyridine (4,4'-DTDP, 1 mm), as well as the hydrophilic thimerosal (1 mm), activated and then inhibited RyRs from either the cis (cytoplasmic) or trans (luminal) solutions. Activation was associated with an increase in the (a) mean channel open time and (b) number of exponential components in the open time distribution from one ( approximately 2 msec) to three (approximately 1 msec; approximately 7 msec; approximately 15 msec) in channels activated by trans 4,4'-DTDP or cis or trans thimerosal. A longer component (approximately 75 msec) appeared with cis 4, 4'-DTDP. Activation by either oxidant was reversed by the thiol reducing agent, dithiothreitol. The results suggest that three classes of cysteines are available to 4,4'-DTDP or thimerosal, SHa or SHa* activating the channel and SHi closing the channel. SHa is either distributed over luminal and cytoplasmic RyR domains, or is located within the channel pore. SHi is also located within the transmembrane domain. SHa* is located on the cytoplasmic domain of the protein.

Activation of the cardiac ryanodine receptor by sulfhydryl oxidation is modified by Mg2+ and ATP.

The reactive disulfide 4,4'-dithiodipyridine (4,4' DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca2+] of 10(-7) M (in the absence of Mg2+ and ATP), increased within approximately 1 min of addition of 1 mM 4,4'-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mM cis Mg2+ (10(-7) M cis Ca2+) or by 10 mM cis Mg2+ (10(-3) M cis Ca2+), or activated by 4 mM ATP (10(-7) M cis Ca2+), also responded to 1 mM cis 4,4'-DTDP with activation and then loss of activity. Po and mean open time (T(o)) of the maximally activated channels were lower in the presence of Mg2+ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mM 4,4'-DTDP in channels inhibited by Mg2+, and the previously reported enhanced activation by 4,4'-DTDP in channels activated by Ca2+ or caffeine (Eager et al., 1997), the activation produced by 1 mM cis 4,4'-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation.

Actions of sulfhydryl reagents on single ryanodine receptor Ca(2+)-release channels from sheep myocardium.

Effects of the reactive disulfides, 2,2'- and 4,4'-dithiodipyridine, on single cardiac ryanodine receptor (RyR) ion channels incorporated into lipid bilayers are reported. RyRs are activated within minutes of addition of the reactive disulfides (10(-7) to 10(-3) M) with an irreversible loss of channel activity after the activation at concentrations > or = 10(-4) M. This activation, followed by loss of activity, is seen over a wide range of cytoplasmic (cis) Ca2+ concentration between 10(-9) and 2 x 10(-2) M and occurs more rapidly with higher reactive disulfide concentrations or when RyRs are initially active at 10(-5) or 10(-3) M Ca2+. The reactive disulfides increase the channel open probability by introducing long components into the open time distributions, increasing the mean channel open time by up to 50-fold. Closed time distributions are not altered by the sulfhydryl reagents. The effects of the reactive disulfides are prevented by the reducing agents dithiothreitol and glutathione (1-10 mM). The results suggest that cysteine residues on the RyR complex can regulate the ion channel gating mechanisms.

Ion channels in the sarcoplasmic reticulum of striated muscle.

This review provides a summary of current concepts about the structure and single-channel properties of ryanodine receptor calcium release channels and counter ion channels that facilitate Ca2+ release and reuptake by the sarcoplasmic reticulum. Some recent results, obtained with single ryanodine receptor ion channels incorporated into lipid bilayers from terminal cisternae vesicles of rabbit skeletal muscle and sheep ventricular myocardium, are described. The ryanodine receptor is the major Ca2+ release channel in skeletal and cardiac muscle and has been studied in far greater detail than other sarcoplasmic reticulum ion channel proteins. Several ryanodine receptor genes have been cloned and sequenced, and isoforms of the protein have been detected in muscle and in endoplasmic reticulum of brain and many other tissues from mammals, lower vertebrates, nematodes and drosophila. The proteins from all species are tetramers of a peptide with a molecular mass of approximately equal to 560 kDa, containing approximately equal to 5000 amino acids, with a similar maximum single-channel conductance of 500-800 row S for monovalent cations at 250mM. Results presented here include: Ca2+ activation and adaptation of activity in skeletal ryanodine receptors with rapid changes in [Ca2+] controlled by perfusion; activation by FK506 and regulation of cooperative gating of skeletal ryanodine receptor channel activity by FK506-binding proteins; activation and block of cardiac ryanodine receptors by addition of reactive disulphides and by bilayer voltage. Effects of phosphorylation, calmodulin, triadin, calsequestrin and interactions with the alpha 1 subunit of the dihydropyridine receptor on ryanodine receptor activity are summarized. Potassium and chloride channels in skeletal muscle sarcoplasmic reticulum, are described.

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle.

Ca(2+)-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca2+ inhibited cardiac RyRs with an average Km = 15 mM, skeletal RyRs with Km = 0.7 mM and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca2+ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [3H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca(2+)-inhibition in cardiac RyRs (Chu, Fill, Stefani & Entman (1993) J. Membrane Biol. 135, 49-59). Ryanodine prevented Ca2+ from inhibiting either cardiac or skeletal RyRs. Ca(2+)-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mM Cs+, but not by 500 mM K+, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca(2+)-activation. Ca(2+)-inhibition in skeletal RyRs was retained in the presence of 500 mM Cs+. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mM Cs+ in the solutions fail to demonstrate Ca(2+)-inhibition, while Ca(2+)-inhibition of Ca2+ release is observed in vesicle studies where K+ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca(2+)-inhibition in skeletal RyRs and cardiac RyRs, with different Ca2+ affinities for inhibition. We conclude that differences in the Ca(2+)-inhibition in cardiac and skeletal channels depends on their Ca2+ binding properties.

Endogenous opioid modulation of hypercapnic-stimulated respiration in the rat.

The role of endogenous opioids in respiratory control in the pentobarbital anaesthetised rat was investigated using a rebreathing technique to generate a progressively increasing hypercapnic stimulus to the respiratory centers following administration of an opioid antagonist or agonist. Respiratory output was measured by intraesophageal pressure (IEP) changes, and a ventilatory equivalent (VEq) was calculated by multiplying IEP by respiratory rate (mmHg.min-1). A non-selective opioid antagonist, naloxone (0.4 mg/kg i.v.), significantly enhanced the slope of the CO2 response curve for VEq (20 +/- 3 mmHg.min-1.%CO2-1) compared with the control (14 +/- 2 mmHg.min-1.%CO2(-1)) (P < 0.05; n = 14). A similar enhancement of the hypercapnic response by naloxone was found in rats anaesthetised with urethane (n = 5). The mu receptor agonist dermorphin (1 mg/kg i.v.) significantly depressed the slope of the CO2 response curve for IEP (-0.01 +/- 0.03) compared with the control (0.10 +/- 0.03) in pentobarbital anaesthetised rats (P < 0.05; n = 5) but had no significant effect on respiratory rate. These results suggest a role of endogenous opioids in the modulation of respiration during hypercapnia.