Pauline R. Junankar

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

The immunosuppressant drug FK‐506 (3–20 μM) 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 [15].

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.

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.

Metabolite profiling of the intraerythrocytic malaria parasite Plasmodium falciparum by (1)H NMR spectroscopy.

NMR spectroscopy was used to identify and quantify compounds in extracts prepared from mature trophozoite‐stage Plasmodium falciparum parasites isolated by saponin‐permeabilisation of the host erythrocyte. One‐dimensional 1H NMR spectroscopy and four two‐dimensional NMR techniques were used to identify more than 50 metabolites. The intracellular concentrations of over 40 metabolites were estimated from the 1H NMR spectra of extracts prepared by four extraction methods: perchloric acid, methanol/water, methanol/chloroform/water, and methanol alone. The metabolites quantified included: the majority of the biological α‐amino acids; 4‐aminobutyric acid; mono‐, di‐ and tri‐carboxylic acids; nucleotides; polyamines; myo‐inositol; and phosphocholine and phosphoethanolamine. The parasites also contained a significant concentration (up to 12 mM) of the exogenous buffering agent, HEPES. Although the metabolite profiles obtained with each extraction method were broadly similar, perchloric acid was found to have significant advantages over the other extraction media. Copyright

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.

The Role of P2Y1 Purinergic Receptors and Cytosolic Ca2+ in Hypotonically Activated Osmolyte Efflux from a Rat Hepatoma Cell Line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca2+ concentration ([Ca2+] i ) to increase transiently by ∼90 nm. This rise in [Ca2+] i was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca2+] i increase. The hypotonically induced rise in [Ca2+] i was prevented by the prior discharge of the intracellular Ca2+ store of the cells by thapsigargin. Removal of extracellular Ca2+ or inhibition of Ca2+ influx by 1–10 μm Gd3+depleted the thapsigargin-sensitive Ca2+ stores and thereby diminished the rise in [Ca2+] i . The hypotonically induced rise in [Ca2+] i was prevented by adenosine 2′-phosphate-5′-phosphate (A2P5P) and pyridoxyl-5′-phosphate-6-azophenyl-2′,4′-disulfonate, inhibitors of purinergic P2Y1 receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca2+] i elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca2+] i elicited by the addition of the P2Y2 and P2Y4 receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y1, P2Y2, and P2Y6 receptors. Inhibition of the hypotonically induced rise in [Ca2+] i blocked hypotonically induced K+ (86Rb+) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl− (125I−) efflux. The interaction of extracellular ATP and/or ADP with P2Y1purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.

Porin-type1 proteins in sarcoplasmic reticulum and plasmalemma of striated muscle fibres

SummaryThe location of porin-type1 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 (Mr) 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 μg of membrane protein blotted. Contamination of the sacroplasmic 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.

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.

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 μm) activated six of eight native, four of four control‐incubated and eleven of eleven FKBP12‐‘stripped’ RyR channels. Midecamycin (cis, 10–30 μm) 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 μm ivermectin or midecamycin, showed normal amounts of FKBP12. In contrast, no FKBP12 was detected after incubation with 40 μm rapamycin. 4 Ivermectin reduced Ca2+ uptake by the SR Ca2+‐Mg2+‐ATPase. Ca2+ uptake by TC fell to ∼40% in the presence of ivermectin (10 μm), both with and without 10 μm Ruthenium Red. Ca2+ uptake by longitudinal SR also fell to ∼40% with 10 μm ivermectin. Midecamycin (10 μm) reduced Ca2+ uptake by TC vesicles to ∼76% without Ruthenium Red and to ∼90% with Ruthenium Red. 5 The rate of rise of extravesicular [Ca2+] increased ∼2‐fold when 10 μm 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 ∼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 ∼90%, and reversibly increased submaximal caffeine‐induced contraction in five of eight fibres by ∼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.

Osmotic swelling activates two pathways for K+ efflux in a rat hepatoma cell line.

The pathways for the efflux of K⁺ from osmotically-swollen HTC rat hepatoma cells were investigated using ⁸⁶Rb⁺ as a tracer for K⁺. Exposure of HTC cells to a hypotonic solution (< 250 mOsm kg^-1) resulted in a transient efflux of ⁸⁶Rb⁺ that reached a maximal value after ñ1 min, and inactivated within 3 min. This initial ⁸⁶Rb⁺ efflux was inhibited by charybdotoxin, clotrimazole and Ba²⁺, but not by apamin or paxilline, consistent with it being via an intermediate-conductance Ca²⁺-activated K⁺ channel. For cells exposed to an extracellular osmolality < 180 mOsm kg^-1 there was an additional ⁸⁶Rb⁺ efflux component which was slower to activate, taking 4 - 6 min to reach a maximum, and remaining active for > 20 min. The second ⁸⁶Rb⁺ efflux component was not inhibited by K⁺ channel blockers but was inhibited by the anion channel blockers, tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumate. The time-courses for its activation and inactivation, as well as its dependence on the extracellular osmolality, were very similar to those observed for the hypotonically-activated efflux of the organic osmolyte, taurine. The data are consistent with the second component of ⁸⁶Rb⁺ efflux and the efflux of taurine from osmotically-swollen cells occurring via a common pathway having a marked selectivity for taurine over ⁸⁶Rb⁺.