Biomedical subjects

A F Dulhunty

Publications and source records attributed to A F Dulhunty.

At least 19 recordsLinked to original sources

PubMed2001

Phosphate ion channels in sarcoplasmic reticulum of rabbit skeletal muscle.

1. Phosphate ions (P(i)) enter intracellular Ca2+ stores and precipitate Ca2+. Since transport pathways for P(i) across the membrane of intracellular calcium stores have not been identified and anion channels could provide such a pathway, we have examined the P(i) conductance of single anion channels from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle using the lipid bilayer technique. 2. Two anion channels in skeletal muscle SR, the small conductance (SCl) and big conductance (BCl) chloride channels, were both found to have a P(i) conductance of 10 pS in 50 mM P(i). The SCl channel is a divalent anion channel which can pass HPO4(2-) as well as SO4(2-) (60 pS in 100 mM free SO4(2-)). The BCl channel is primarily a monovalent anion channel. The SCl and BCl channels are permeable to a number of small monovalent anions, showing minor selectivity between Cl-, I- and Br- (Cl- > I- > Br-) and relative impermeability to cations and large polyatomic anions (Cs+, Na+, choline+, Tris+, Hepes- and CH3O3S-). 3. The P(i) conductance of SCl and BCl channels suggests that both channel types could sustain the observed P(i) fluxes across the SR membrane. Comparison of the blocking effects of the phosphonocarboxylic acids, ATP and DIDS, on the anion channels with their effects on P(i) transport suggests that the SCl channel is the more likely candidate for the SR P(i) transport mechanism. 4. The SCl channel, with previously unknown function, provides a regulated pathway for P(i) across the SR membrane which would promote P(i) entry and thereby changes in the rapidly releasable Ca2+ store during onset and recovery from muscle fatigue. Anion channels may provide a pathway for P(i) movement into and out of Ca2+ stores in general.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2001

Characteristics of irreversible ATP activation suggest that native skeletal ryanodine receptors can be phosphorylated via an endogenous CaMKII.

Phosphorylation of skeletal muscle ryanodine receptor (RyR) calcium release channels by endogenous kinases incorporated into lipid bilayers with native sarcoplasmic reticulum vesicles was investigated during exposure to 2 mM cytoplasmic ATP. Activation of RyRs after 1-min exposure to ATP was reversible upon ATP washout. In contrast, activation after 5 to 8 min was largely irreversible: the small fall in activity with washout was significantly less than that after brief ATP exposure. The irreversible activation was reduced by acid phosphatase and was not seen after exposure to nonhydrolyzable ATP analogs. The data suggested that the channel complex was phosphorylated after addition of ATP and that phosphorylation reduced the RyR's sensitivity to ATP, adenosine, and Ca(2+). The endogenous kinase was likely to be a calcium calmodulin kinase II (CaMKII) because the CaMKII inhibitor KN-93 and an inhibitory peptide for CaMKII prevented the phosphorylation-induced irreversible activation. In contrast, phosphorylation effects remained unchanged with inhibitory peptides for protein kinase C and A. The presence of CaMKIIbeta in the SR vesicles was confirmed by immunoblotting. The results suggest that CaMKII is anchored to skeletal muscle RyRs and that phosphorylation by this kinase alters the enhancement of channel activity by ATP and Ca(2+).

Acid Phosphatase↗

PubMed2001

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop.

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.

Amino Acid Sequence↗

PubMed2001

Structural determinants for activation or inhibition of ryanodine receptors by basic residues in the dihydropyridine receptor II-III loop.

The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr671- Leu690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at -40 mV), was seen with peptides containing the basic residues 681Arg Lys Arg Arg Lys685, and was strongest when the residues were a part of an alpha-helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor.

Amino Acid Sequence↗

PubMed2000

Skeletal muscle ryanodine receptor channels are activated by the fungal metabolite, gliotoxin.

Interactions between the reactive disulfide fungal metabolite, gliotoxin (GTX), and rabbit skeletal ryanodine receptor (RyR) calcium release channels have been examined. RyRs in terminal cisternae vesicles formed a covalent complex with 100 microm (35)S-GTX, which was reversed by 1 mm dithiothreitol (DTT) or 1 mm glutathione. GTX (80-240 microm), added to either cytoplasmic (cis) or luminal (trans) solutions, increased the rate of Ca(2+) release from SR vesicles and the frequency of opening of single RyR channels in lipid bilayers. Channel activation was reversed upon addition of 2 mm DTT to the cis solution, showing that the activation was due to an oxidation reaction (2 mm DTT added to the cis solution in the absence of GTX did not affect RyR activity). Furthermore, RyRs were not activated by trans GTX if the cis chamber contained DTT, suggesting that GTX oxidized a site in or near the membrane. In contrast to cis DTT, 2 mm DTT in the trans solution increased RyR activity when added either alone or with 200 microm trans GTX. The results suggest that (i) GTX increases RyR channel activity by oxidizing cysteine residues that are close to the membrane and located on RyR, or associated proteins, and (ii) a disulfide bridge or nitrosothiol, accessible only from the luminal solution, normally suppresses RyR channel activity. Some of the actions of GTX in altering Ca(2+) homeostatsis might depend on its modification of RyR calcium channels.

Animals↗

PubMed2000

A structural requirement for activation of skeletal ryanodine receptors by peptides of the dihydropyridine receptor II-III loop.

The solution structures of three related peptides (A1, A2, and A9) corresponding to the Thr(671)-Leu(690) region of the skeletal muscle dihydropyridine receptor II-III loop have been investigated using nuclear magnetic resonance spectroscopy. Peptide A1, the native sequence, is less effective in activating ryanodine receptor calcium release channels than A2 (Ser(687) to Ala substitution). Peptide A9, Arg(681)-Ser(687), does not activate ryanodine receptors. A1 and A2 are helical from their N terminus to Lys(685) but are generally unstructured from Lys(685) to the C terminus. The basic residues Arg(681)-Lys(685), essential for A1 activation of ryanodine receptors, are located at the C-terminal end of the alpha-helix. Peptide A9 was found to be unstructured. Differences between A1 and A2 were observed in the C-terminal end of the helix (residues 681-685), which was less ordered in A1, and in the C-terminal region of the peptide, which exhibited greater flexibility in A1. Predicted low energy models suggest that an electrostatic interaction between the hydroxyl oxygen of Ser(687) and the guanidino moiety of Arg(683) is lost with the Ser(687)Ala substitution. The results show that the more structured peptides are more effective in activating ryanodine receptors.

Amino Acid Sequence↗

PubMed2000

Nitric oxide activates or inhibits skeletal muscle ryanodine receptors depending on its concentration, membrane potential and ligand binding.

We show that rabbit skeletal RyR channels in lipid bilayers can be activated or inhibited by NO, in a manner that depends on donor concentration, membrane potential and the presence of channel agonists. 10 microm S-nitroso-N-acetyl-penicillamine (SNAP) increased RyR activity at -40 mV within 15 sec of addition to the cis chamber, with a 2-fold increase in frequency of channel opening (F(o)). 10 microm SNAP did not alter activity at +40 mV and did not further activate RyRs previously activated by 2 mm cis ATP at +40 or -40 mV. In contrast to the increase in F(o) with 10 microm SNAP, 1 mm SNAP caused a 2-fold reduction in F(o) but a 1.5-fold increase in mean open time (T(o)) at -40 mV in the absence of ATP. 1 mm SNAP or 0.5 mm sodium nitroprusside (SNP) induced approximately 3-fold reductions in F(o) and T(o) at +40 or -40 mV when channels were activated by 2 mm cis ATP or in channels activated by 6.5 microm peptide A at -40 mV (peptide A corresponds to part of the II-III loop of the skeletal dihydropyridine receptor). Both SNAP-induced activation and SNAP/SNP-induced inhibition were reversed by 2 mm dithiothreitol. The results suggest that S-Nitrosylation or oxidation of at least three classes of protein thiols by NO each produced characteristic changes in RyR activity. We propose that, in vivo, initial release of NO activates RyRs, but stronger release increases [NO] and inhibits RyR activity and contraction.

Adenosine Triphosphate↗

PubMed2000

Cadmium withdrawal contractures in rat soleus muscle fibres.

Transient "Cd2+ withdrawal" contractures, with amplitudes of < or =60% peak tetanic tension, were seen when > or =3 mM Cd2+ was removed, after exposures lasting > or =5 min, from solutions bathing rat soleus fibres at 22 degrees C with Cl- as the major external anion. The minimum free [Cd2+] for withdrawal contractures was reduced twofold when the external anion was SO4(2-). Withdrawal contractures were not seen after removal of 3 mM Co2+, Zn2+ or La3+ and were not observed in rat extensor digitorum longus fibres. The contractures were not due to depolarization (membrane potential, Vm, did not change during Cd2+ removal) or to an influx of external Ca2+ (the transient tension increase was recorded when solutions either lacked Ca2+, or contained 2 mM Co2+). Cd2+ withdrawal contractures were abolished by inactivation of excitation-contraction coupling (ECC) following depolarization in 40 mM K+ for 20 min, and recovered from inactivation at the same time as twitch and tetanic contractions with repriming of ECC. Withdrawal contractures were depressed by agents that depress ECC, i.e. low [Ca2+], 2 mM Co2+, 30 mM Ca2+, 30 mM Mg2+ and 50 microM nifedipine. The results support a hypothesis in which withdrawing Cd2+ from the external solution induces a contracture by activating the voltage sensor for ECC.

Animals↗

PubMed2000

Delayed contractures induced by external cadmium ions in rat soleus muscle fibres.

Cd(2+)-induced contractures began with a delay of approximately =4 min after adding 3 mM Cd2+ to external solutions that contained Cl- as the major anion. Tension increased to approximately =20% of peak tetanic tension after 30 min and was maintained after Cd2+ washout. Tension developed more rapidly at higher [Cd2+] (up to 10 mM). There was a lack of correlation between the delay before the contracture and contracture tension: (1) tension was reduced by 2 mM CO2+ or 50 microM nifedipine, although the delay remained at approximately =4 min, and (2) the delay fell to seconds when Cd2+ was added in SO42- solutions, although tension was the same as in Cl- solutions. Since (SO4)2- solutions swell T-tubules, Cd2+ may enter the T-system before inducing contractures. Cd(2+)-induced contractures depended on external [Ca2+] since they were reduced when Ca2+ was omitted from solutions. The contractures did not depend on activation of excitation-contraction coupling, since tension was not altered when the voltage sensor was inactivated by depolarization in 40 mM K+. A small contracture developed with 3 mM Zn2+, but not 3 mM Co2+ or La3+. Both Cd2+ and Zn2+ activated the contractile proteins in skinned fibres. Cd(2+)-induced contractures may depend on external Cd2+ releasing Ca2+ from the sarcoplasmic reticulum (SR), or on Cd2+ entering the fibre, releasing Ca2+ from the SR and/or directly activating the contractile proteins.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Effects of external cadmium ions on excitation-contraction coupling in rat soleus fibres.

The actions of external Cd2+ on the twitch and tetanic contractions, action potentials and potassium (K+) contractures of rat soleus muscle fibre bundles have been investigated. Cd2+ at 1-1.5 mM did not significantly alter tetanic tension, but increased twitch tension and increased the duration and overshoot of action potentials. At >/=3 mM, Cd2+ (1) depressed tetanic contractions and initially potentiated but later depressed twitches, (2) abolished the action potential overshoot, and (3) shifted peak K+ contracture tension to more positive membrane potentials. Twitch and tetanic contractions, and action potentials remained depressed when Cd2+ was washed out of the bath. The effects of Cd2+ on the twitch, tetanus and action potential were mimicked by Zn2+, while La3+ and Co2+ at 3 mM - or Mg2+ and Ca2+ at 30 mM - depressed peak twitch and tetanic tension, but did not potentiate twitches. The results suggest that: (1) Cd2+ and Zn2+ potentiate twitch tension by prolonging action potential depolarisation; (2) Cd2+ depresses twitch and tetanic tension by reducing the action potential overshoot, and causing a positive shift in the voltage dependence of contraction; and (3) the irreversible depression of action potential amplitude in rat soleus muscle is a specific property of Cd2+ and Zn2+ that is not shared by Co2+, Mg2+ or Ca2+.

Action Potentials↗

PubMed1999

Activation and inhibition of skeletal RyR channels by a part of the skeletal DHPR II-III loop: effects of DHPR Ser687 and FKBP12.

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2 > A1 > A1S > NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by approximately 30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at -40 mV, with 10 microM A1 or 50 nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100 nM cis Ca2+. Inhibition by A1 was greatest at +40 mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100 microM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.

Adenosine Triphosphate↗

PubMed1999

Oxidation and reduction of pig skeletal muscle ryanodine receptors.

Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615)) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents 4,4'-dithiodipyridine (4, 4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all reagents. DTNB (1 mM), either cytoplasmic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB, inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTDP and DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys(615) in RyR(MH) is functionally unimportant in redox cycling.

Animals↗

PubMed1998

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.

Adenosine Triphosphate↗

PubMed1997

Inositol polyphosphates modify the kinetics of a small chloride channel in skeletal muscle sarcoplasmic reticulum.

The actions of D-myo-inositol 1,4,5-trisphosphate (IP3) and D-myo-inositol 1,3,4,5-tetrakisphospate (IP4) on small chloride (SCl) channels from rabbit skeletal muscle sarcoplasmic reticulum are reported. We find that the inositol polyphosphates (6-40 microM) are potent reversible blockers of SCl channels in lipid bilayers at -40 mV with >10(-5) m cis (cytoplasmic) Ca2+ when added to the cis, but not trans, chamber. IP3 or IP4 at 20 microM reduced the mean open time from 89 +/- 16 msec to 11 +/- 2 msec or to 8.0 +/- 1.0 msec respectively, by abolishing the longest time constant component in the open time distribution. Neither IP3 nor IP4 altered the six single-channel conductance levels. The fraction of low conductance events increased approximately 4-fold and the dwell time at the lower conductance levels increased approximately 3-fold. Channel gating was altered so that most transitions were between the closed level and an open level, in contrast to control channels which remained open for long periods with many transitions between the six open levels. The actions of the inositol polyphosphates were: (1) not prevented by 20 microg/ml cis heparin (an IP3 receptor blocker); (2) mimicked by 10 microM cis synthetic inositol polyphosphates, L-chiro-inositol 1,4,6-trisphosphate and L-chiro-inositol 1,4,6-trisphosphorothionate (which do not bind to IP3 receptors); (3) mimicked by cis additions of the polyanions heparin or hepran (20 microg/ml each) and vanadate (500 microM). The results suggest that an interaction between polyanions and SCl channels would allow the channels to be modulated in vivo by inositol polyphosphates.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms.

The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca2+ and Mg2+ concentrations. RyRs from skeletal and cardiac muscle are activated by microm Ca2+ and inhibited by mm Ca2+ and Mg2+. 45Ca2+ release from skeletal SR vesicles suggests two mechanisms for Mg2+-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236-244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg2+- and Ca2+-dependent gating kinetics confirm that there are two mechanisms for Mg2+ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg2+ reduces Po by competing with Ca2+ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca2+ and Mg2+ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg2+ effect depend on cytoplasmic [Ca2+] in such a way that Mg2+ inhibition has the properties of Types-I and II inhibition at low and high [Ca2+] respectively. Both mechanisms are equally important when [Ca2+] = 10 microm in cardiac RyRs or 1 microm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg2+ inhibition, as is often assumed, and we discuss the physiological implications of this finding.

Animals↗