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.

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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.

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.

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 (1)H 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 (1)H 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 alpha-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.

The immunosuppressant drug 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 Ca 2+ inactivation of the ryanodine receptor seen at 10 -3 M Ca 2+. The results are consistent with FK-506 removal of FK-506 binding protein from the ryanodine receptor [15].

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.

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