Temperature and pH dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoproteins.

Influenza virus (strain X-47) was labeled with the triplet probe, eosin 5-isothiocyanate. Most of the label was found to be associated with haemagglutinin, the major glycoprotein of the viral envelope. Rotational diffusion of the glycoprotein was investigated by measuring flash-induced transient dichroism of the eosin probe. The anisotropy decay curves showed that mobility of haemagglutinin measured at pH 7.3 increased considerably with temperature with the greatest change occurring over the range 20-30 degrees C. However, at pH 5.2 no mobility was detectable over the time range of the experiment. The activity of the virus was determined by assaying haemolysis of human erythrocytes. The haemolytic activity showed an optimum at pH 5.2 and increased markedly with temperature, being negligible below 20 degrees C. In addition, inactivation of the virus by incubation at pH 5.2 was also strongly temperature dependent. A 15 min incubation at pH 5.2 inactivated the virus above 30 degrees C but had no effect below 20 degrees C. On the basis of these results, it is proposed that mobility of haemagglutinin is significant for its functional properties. When the pH is reduced from 7.3 to 5.2, the mobility observed at higher temperatures is required for the molecular rearrangements which accompany the fusion event. In the absence of an apposing membrane, these rearrangements result in irreversible aggregation of haemagglutinin in the viral membrane, and hence loss of mobility and activity.

Single channel activity of the ryanodine receptor calcium release channel is modulated by FK-506.

The immunosuppressant drug FK-506 (3-20 microM) increased the open probability of ryanodine receptor calcium release channels, formed by incorporation of terminal cisternae vesicles from rabbit skeletal muscle into lipid bilayers, with cis (cytoplasmic) calcium concentrations between 10(-7) M and 10(-3) M. FK-506 increased mean current and channel open time and induced long sojourns at subconductance levels that were between 28% and 38% of the maximum conductance and were distinct from the ryanodine-induced subconductance level at about 45% of the maximum conductance. FK-506 relieved the Ca2+ inactivation of the ryanodine receptor seen at 10(-3) M Ca2+. The results are consistent with FK-506 removal of FK-506 binding protein from the ryanodine receptor.

Distribution of ryanodine receptor-like immunoreactivity in mammalian central nervous system is consistent with its role in calcium-induced calcium release.

The distributions of ryanodine receptor-like immunoreactivity and Ca-ATPase-like immunoreactivity were identified in the guinea-pig and rat central nervous system using antibodies raised against the rabbit skeletal muscle ryanodine receptor and Ca-ATPase. In both guinea-pig and rat cerebellum, the ryanodine receptor-like immunoreactivity was restricted to the soma and dendrites of Purkinje cells. In the medulla, neuron somata in the hypoglossal nucleus were stained in both species, but in the dorsal motor nucleus of the vagus somata were stained in guinea-pigs but not in rats. This species difference in ryanodine receptor-like immunoreactivity is consistent with the species difference in expression of a ryanodine sensitive, calcium activated potassium conductance in neurons of the dorsal motor nucleus of the vagus. Immunoreactivity to Ca-ATPase was present in vagal motoneurons in both species with denser staining in the guinea-pig. The data further support the idea that, in neurons of the dorsal motor nucleus of the vagus, release of intracellular calcium stores via a ryanodine receptor activates a specific class of potassium channels, thereby modulating cell excitability.

Immunogold labeling of calcium ATPase in sarcoplasmic reticulum of skeletal muscle: use of 1-nm, 5-nm, and 10-nm gold.

We evaluated the use of immunogold electron microscopy to study the distribution of calcium ATPase in the sarcoplasmic reticulum membrane of skeletal muscle. We examined (a) 1-nm gold labeling, (b) the effect of gold size on immunolabeling, and (c) the densities of gold particles in areas of maximal labeling in fibers from rat extensor digitorum longus and pig gracilis muscles. The technique allowed unequivocal identification of the calcium ATPase. Gold particles of 1 nm were successfully visualized in unstained or lightly stained sections and the density of labeling was about 20 times greater than with 10-nm gold. The average densities in areas of intense labeling were 2878 +/- 139/microns 2 with 5-nm gold and 4310 +/- 276/microns 2 with 1-nm gold. These numbers are similar to the density of particles in freeze-fracture replicas of sarcoplasmic reticulum. The low density of 10-nm gold suggests that the large gold particles hinder binding of secondary to primary antibodies. The difference between 1- and 5-nm gold is explained by the amounts of gold conjugated to the immunoglobulin. The results suggest that there is a one-to-one relationship between secondary immunoglobulins (1-nm or 5-nm gold conjugates) and oligomeric complexes of calcium ATPase.

Extra-junctional ryanodine receptors in the terminal cisternae of mammalian skeletal muscle fibres.

The distribution of ryanodine receptor calcium-release channels over the terminal cisternae (TC) membrane of skeletal muscle fibres was examined by using immunogold electron microscopy. Two monoclonal antibodies (5C3 and 8E2) that bound to monomers of the ryanodine receptor protein on Western blots of SDS-polyacrylamide gels were used to locate calcium-release channels in longitudinal sections of rat sternomastoid and diaphragm fibres. Up to 21% of 5C3 binding on TC membranes was extra-junctional, compared with 46% for 8E2. Binding of 8E2 to the fibres was less than half that of 5C3, possibly because of steric shielding of the 8E2 antigenic site at the junction. The distances between neighbouring particles in clusters was 20-40 nm, i.e. the distance between subunits of the ryanodine receptor or between neighbouring foot structures. We suggest that, during activation, extra-junctional ryanodine receptors may release Ca2+ directly into the myoplasm, rather than into the restricted space of the triad junction.

Calcium ATPase in the sarcoplasmic reticulum of muscle from normal and malignant hyperthermia susceptible pigs.

The density of Mg(2+)-dependent Ca2+ ATPase in the terminal cisternae of pig skeletal muscle fibers was investigated to discover whether a reduction in Ca2+ ATPase content impairs Ca2+ sequestration and contributes to the elevated myoplasmic Ca2+ concentration in malignant hyperthermia. Unexpectedly, immunogold electron microscopy showed an increase in Ca2+ ATPase, while densitometry of SDS-polyacrylamide gels suggested that the Ca2+ ATPase content of terminal cisternae vesicles did not change. The affinity of Ca2+ ATPase in vesicles for our monoclonal antibody was not altered. We suggest that the availability of antigenic sites in malignant hyperthermia increases after processing for electron microscopy, perhaps as a consequence of altered sarcoplasmic reticulum membrane properties.

Subcellular distribution of ryanodine receptor-like and calcium ATPase-like immunoreactivity in brainstem and cerebellar neurones of rat and guinea pig.

Monoclonal antibodies against ryanodine receptor (5C3) and calcium ATPase (D12) of skeletal muscle sarcoplasmic reticulum were used in an immunoelectron microscopic study of cerebellar Purkinje cells and neurons of the hypoglossal and dorsal motor nuclei of the vagus (DMV) from rat and guinea-pig. All neurones were labelled with D12 and all, except rat DMV, labelled with 5C3. Most labelling was on smooth endoplasmic reticulum within 500 nm of the plasmalemma where Ca(2+)-activated Ca2+ release would rapidly increase cytosolic calcium following a small Ca2+ influx across the plasmalemma.

Ryanodine receptors from rabbit skeletal muscle are reversibly activated by rapamycin.

In this report we demonstrate that the immunosuppressive drug, rapamycin, can reversibly activate the skeletal muscle ryanodine receptor calcium release channel (RyR) in terminal cisternae vesicles incorporated into planar lipid bilayers. This reveals a second mechanism of activation of RyRs by rapamycin. Irreversible channel activation and openings to subconductance levels are seen when rapamycin forms a complex with and removes the tightly bound 12 kDa FK506-binding protein (FKBP12) from the RyR. We show here that micromolar rapamycin activates RyRs which were previously 'stripped' of > 95% of their FKBP12s. Rapamycin caused a 6-fold increase in mean current, which was largely reversible, but no increase in the fraction of openings to subconductance levels. Therefore native RyRs, stripped of FKBP12, are directly activated by the macrocyclic lactone, rapamycin.

Monoclonal antibody to skeletal muscle ryanodine receptor detects a polypeptide in rat brain: comparison of immunogenic fragments after limited proteolysis.

A polypeptide of high molecular mass has been detected in mammalian brain by a monoclonal antibody, 5C3, raised against skeletal muscle ryanodine receptor. 5C3 does not crossreact with the cardiac ryanodine receptor, the isoform which is believed to be located in many regions of the brain. Endogenous proteases in brain formed a prominent immunogenic fragment of 116 kDa whereas five immunostaining polypeptides greater than 200 kDa were observed in skeletal muscle. Mild trypsin digestion of brain microsomes resulted in fragments of approximately 400 and approximately 280 kDa, of similar mass to two peptides formed from the skeletal muscle ryanodine receptor. However a peptide of 28 kDa, resistant to trypsin, was observed in muscle but not in brain. The brain polypeptide recognised by 5C3 is therefore not identical to the skeletal muscle ryanodine receptor.

Organic osmolyte channels: a comparative view.

Cells respond to osmotic swelling by releasing inorganic ions and small organic molecules (organic osmolytes). In many cell-types, osmotic swelling results in the activation of an outwardly-rectifying anion-selective current. The channel underlying this current has a significant permeability to a number of organic osmolytes and may play a role in the hypoosmotically-activated efflux of these compounds. However, there is also evidence that the volume-regulatory efflux of organic osmolytes involves other pathways which may be selective for neutral osmolytes over anions.

Sequence studies of rabbit serum transferrin.

Large fragments of rabbit serum transferrin have been prepared by enzymatic digestion with subtilisin and by chemical cleavage with BNPS-skatole. Sequence determinations on fragments from the N-terminal lobe lead to the assignment of 273 residues and those from the C-terminal lobe 267 residues. Together with previous determinations, a total of 614 of the ca 679 residues in rabbit transferrin have been assigned. A number of corrections are made to the preliminary sequence assignments of O.U. Beg, H.A. McKenzie and D.C. Shaw (1988) Biochemistry International 17, 1135-1142.

Subconductance states in single-channel activity of skeletal muscle ryanodine receptors after removal of FKBP12.

FKBP12 was removed from ryanodine receptors (RyRs) by incubation of rabbit skeletal muscle terminal cisternae membranes with rapamycin. The extent of FKBP12 removal was estimated by immunostaining Western blots of terminal cisternae proteins. Single FKBP12-depleted RyR channels, incorporated into planar lipid bilayers, were modulated by Ca2+, ATP, ryanodine, and ruthenium red in the cis chamber and opened frequently to the normal maximum conductance of approximately 230 pS and to substate levels of approximately 0.25, approximately 0.5, and approximately 0.75 of the maximum conductance. Substate activity was rarely seen in native RyRs. Ryanodine did not after the number of conductance levels in FKBP12-depleted channels, but, at a membrane potential of +40 mV, reduced both the maximum and the substate conductances by approximately 50%. FKBP12-stripped channels were activated by a 10-fold-lower [Ca2+] and inhibited by a 10-fold-higher [Ca2+], than RyRs from control-incubated and native terminal cisternae vesicles. The open probability (Po) of these FKBP12-deficient channels was greater than that of control channels at 0.1 microM and 1 mM cis Ca2+ but no different at 10 microM cis Ca2+, where channels showed maximal Ca2+ activation. The approximately 0.25 substate was less sensitive than the maximum conductance to inhibition by Ca2+ and was the dominant level in channels inhibited by 1 mM cis Ca2+. The results show that FKBP12 coordinates the gating of channel activity in control and ryanodine-modified RyRs.

Raised intracellular [Ca2+] abolishes excitation-contraction coupling in skeletal muscle fibres of rat and toad.

1. Raising the intracellular [Ca2+] for 10 s at 23 degrees C abolished depolarization-induced force responses in mechanically skinned muscle fibres of toad and rat (half-maximal effect at 10 and 23 microM, respectively), without affecting the ability of caffeine or low [Mg2+] to open the ryanodine receptor (RyR)/Ca2+ release channels. Thus, excitation-contraction coupling was lost, even though the Ca2+ release channels were still functional. Coupling could not be restored in the duration of an experiment (up to 1 h). 2. The Ca(2+)-dependent uncoupling had a Q10 > 3.5, and was three times slower at pH 5.8 than at pH 7.1. Sr2+ caused similar uncoupling at twenty times higher concentration, but Mg2+, even at 10 mM, was ineffective. Uncoupling was not noticeably affected by removal of ATP or application of protein kinase or phosphatase inhibitors. 3. Confocal laser scanning microscopy showed that the transverse tubular system was sealed in its entirety in mechanically skinned fibres and that its integrity was maintained in uncoupled fibres. Electron microscopy revealed distorted or severed triad junctions and Z-line aberrations in uncoupled fibres. 4. Only when uncoupling was induced at a relatively slow rate (e.g. over 60 s with 2.5 microM Ca2+) could it be prevented by the protease inhibitor leupeptin (1 mM). Immunostaining of Western blots showed no evidence of proteolysis of the RyR, the alpha 1-subunit of dihydropyridine receptor (DHPR) or triadin in uncoupled fibres. 5. Fibres which, whilst intact, were stimulated repeatedly by potassium depolarization with simultaneous application of 30 mM caffeine showed reduced responsiveness after skinning to depolarization but not to caffeine. Rapid release of endogenous Ca2+, or raised [Ca2+] under conditions which minimized the loss of endogenous diffusible myoplasmic molecules from the skinned fibre, caused complete uncoupling. Taken together, these results suggest that Ca(2+)-dependent uncoupling can also occur in intact fibres. 6. This Ca(2+)-dependent loss of depolarization-induced Ca2+ release may play an important feedback role in muscle by stopping Ca2+ release in localized areas where it is excessive and may be responsible for long-lasting muscle fatigue after severe exercise, as well as contributing to muscle weakness in various dystrophies.

Ultrastructure of sarcoballs on the surface of skinned amphibian skeletal muscle fibres.

The formation of sarcoballs on the surface of skinned fibres from semitendinosus muscles of Xenopus laevis, and the sarcoplasmic reticulum content of the structures, have been studied using conventional electron microscopic techniques and immunoelectron microscopy. Examination of the fibres showed many membrane-bound blebs projecting from the surface in areas where vesicles of internal membranes (including sarcoplasmic reticulum, T-tubules and mitochondria) were clustered in interfilament spaces. The blebs varied in size from 1 micron to 150 microns and those with diameters > 10 microns are referred to as sarcoballs. Small blebs were often seen in close association with each other and might have fused during sarcoball formation. The interior of the sarcoball was filled with foam-like material made up of vesicles with diameters of 100 nm to 1.0 microns. The sarcoplasmic reticulum membrane content of the sarcoballs was evaluated using two monoclonal antibodies, one to the Ca2+ ATPase of the sarcoplasmic reticulum and the second to ryanodine receptor calcium release channels in the junctional-face membrane. The antibodies bound to some components of the surface and interior of the sarcoball, but not to mitochondrial-like structures and tubular vesicles. The results show that a large component of the sarcoball and its surface is derived from sarcoplasmic reticulum and suggest that mitochondria and T-tubules might also contribute membranes to the structures. Our hypothesis is that (a) blebs bud out from the surface of the skinned fibre following fusion of internal vesicles that are extruded along interfilament channels during unrestrained contractures, (b) blebs grow into sarcoballs by additional fusion of internal membrane vesicles and fusion of adjacent blebs, and (c) the sarcoball is a foam-like structure composed of bathing medium and membrane lipid (containing membrane proteins).

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.

Reduced inhibitory effect of Mg2+ on ryanodine receptor-Ca2+ release channels in malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.

Porin-type 1 proteins in sarcoplasmic reticulum and plasmalemma of striated muscle fibres.

The location of porin-type 1 proteins in mammalian striated muscle has been assessed using immunogold electron microscopy with an anti-porin 31HL monoclonal antibody as the primary antibody. Gold particles were found on the mitochondrial outer membrane, the sarcoplasmic reticulum and plasmalemma in longitudinal sections of rat and rabbit skeletal muscle and rabbit and sheep cardiac muscle. The relative densities of gold particles in the mitochondrial outer membrane, sarcoplasmic reticulum and plasmalemma were 7:3:1 in white sternomastoid muscle, for example. Skeletal and cardiac sarcoplasmic reticulum vesicles, which had been fractionated by discontinuous sucrose density centrifugation, were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. The anti-porin 31HL monoclonal antibody detected a band of relative molecular mass (M(r)) 31,000 in all muscle sarcoplasmic reticulum vesicle fractions and also in liver mitochondria. The intensity of immunostaining of the sarcoplasmic reticulum fractions was 2.5-10% that of mitochondrial outer membranes per microgram of membrane protein blotted. Contamination of the sarcoplasmic reticulum fractions by mitochondrial outer membrane was < 0.75% as determined from the specific activity of monoamine oxidase. Thus, only a small part of the porin detected in sarcoplasmic reticulum vesicles can be attributed to mitochondrial contamination. These results show that porin-type1 immunoreactivity is not restricted to mitochondria but found in the sarcoplasmic reticulum and plasmalemma of both mammalian skeletal and cardiac muscle.

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.

Characteristics of two types of chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

A comparison is made of two types of chloride-selective channel in skeletal muscle sarcoplasmic reticulum (SR) vesicles incorporated into lipid bilayers. The I/V relationships of both channels, in 250/50 mM Cl- (cis/trans), were linear between -20 and +60 mV (cis potential,) reversed near Ecl and had slope conductances of approximately 250 pS for the big chloride (BCl) channel and approximately 70 pS for the novel, small chloride (SCl) channel. The protein composition of vesicles indicated that both channels originated from longitudinal SR and terminal cisternae. BCl and SCl channels responded differently to cis SO4(2-) (30-70 mM), 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid (8-80 microM) and to bilayer potential. The BCl channel open probability was high at all potentials, whereas SCl channels exhibited time-dependent activation and inactivation at negative potentials and deactivation at positive potentials. The duration and frequency of SCl channel openings were minimal at positive potentials and maximal at -40 mV, and were stationary during periods of activity. A substate analysis was performed using the Hidden Markov Model (S. H. Chung, J. B. Moore, L. Xia, L. S. Premkumar, and P. W. Gage, 1990, Phil. Trans. R. Soc. Lond. B., 329:265-285) and the algorithm EVPROC (evaluated here). SCl channels exhibited transitions between 5 and 7 conductance levels. BCl channels had 7-13 predominant levels plus many more short-lived substates. SCl channels have not been described in previous reports of Cl- channels in skeletal muscle SR.

Effects of ivermectin and midecamycin on ryanodine receptors and the Ca2+-ATPase in sarcoplasmic reticulum of rabbit and rat skeletal muscle.

1. Ryanodine receptor (RyR) Ca2+ channels in the sarcoplasmic reticulum (SR) of skeletal muscle are regulated by the 12 kDa FK506- (or rapamycin-) binding protein (FKBP12). Rapamycin can also activate RyR channels with FKBP12 removed, suggesting that compounds with macrocyclic lactone ring structures can directly activate RyRs. Here we tested this hypothesis using two other macrocyclic lactone compounds, ivermectin and midecamycin. 2. Rabbit skeletal RyRs were examined in lipid bilayers. Ivermectin (cis, 0.66-40 microM) activated six of eight native, four of four control-incubated and eleven of eleven FKBP12-'stripped' RyR channels. Midecamycin (cis, 10-30 microM) activated three of four single native channels, six of eight control-incubated channels and six of seven FKBP12-stripped channels. Activity declined when either drug was washed out. 3. Neither ivermectin nor midecamycin removed FKBP12 from RyRs. Western blots of terminal cisternae (TC), incubated for 15 min at 37 C with 40 microM ivermectin or midecamycin, showed normal amounts of FKBP12. In contrast, no FKBP12 was detected after incubation with 40 microM rapamycin. 4. Ivermectin reduced Ca2+ uptake by the SR Ca2+-Mg2+-ATPase. Ca2+ uptake by TC fell to approximately 40% in the presence of ivermectin (10 microM), both with and without 10 microM Ruthenium Red. Ca2+ uptake by longitudinal SR also fell to approximately 40% with 10 microM ivermectin. Midecamycin (10 microM) reduced Ca2+ uptake by TC vesicles to approximately 76% without Ruthenium Red and to approximately 90 % with Ruthenium Red. 5. The rate of rise of extravesicular [Ca2+] increased approximately 2-fold when 10 microM ivermectin was added to TC vesicles that had been partially loaded with Ca2+ and then Ca2+ uptake blocked by 200 nM thapsigargin. Ivermectin also potentiated caffeine-induced Ca2+ release to approximately 140% of control. These increases in Ca2+ release were not seen with midecamycin. 6. Ivermectin, but not midecamycin, reversibly reduced Ca2+ loading in four of six skinned rat extensor digitorum longus (EDL) fibres to approximately 90%, and reversibly increased submaximal caffeine-induced contraction in five of eight fibres by approximately 110% of control. Neither ivermectin nor midecamycin altered twitch or tetanic tension in intact EDL muscle fibres within 20 min of drug addition. 7. The results confirm the hypothesis that compounds with a macrocyclic lactone ring structure can directly activate RyRs. Unexpectedly, ivermectin also reduced Ca2+ uptake into the SR. These effects of ivermectin on SR Ca2+ handling may explain some effects of the macrolide drugs on mammals.