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The effects of ionic composition and strength on rabbit skeletal muscle Ca 2؉ release channel (ryanodine receptor) activity were investigated in vesicle- 45

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Sequence comparison suggests that the ryanodine receptors (RyRs) have pore architecture similar to that of the bacterial K+ channel KcsA. The lumenal loop linking the two most C-terminal transmembrane spanning segments in the RyRs has a predicted pore helix and an amino acid motif (GGGIG) similar to the selectivity filter (TVGYG) of KcsA identified by x-ray analysis. The RyRs have many negatively charged amino acid residues in the two regions linking the GGGIG motif and predicted pore helix with the two most C-terminal transmembrane spanning segments. We tested the role of these residues by generating single-site mutants, focusing on amino acid residues conserved among the mammalian RyRs. Replacement of two acidic residues immediately after the GGGIG motif in skeletal muscle ryanodine receptor (RyR1-D4899 and -E4900) with asparagine and glutamine profoundly affected ion permeation and selectivity. By comparison, mutagenesis of aspartate and glutamate residues in the putative linker regions showed a K+ conductance and selectivity for Ca2+ compared to K+ (P(Ca)/P(K)) close to wild-type. The results show that the negatively charged carboxyl oxygens of D4899 and E4900 side chains are major determinants of RyR ion conductance and selectivity.

The effects of ruthenium red (RR) on the skeletal and cardiac muscle ryanodine receptors (RyRs) were studied in vesicle-Ca 2؉ The release and sequestration of Ca 2ϩ ions by the sarcoplasmic reticulum (SR), 1 an intracellular membrane compartment, is essential to the process of cardiac and skeletal muscle contraction and relaxation. The rapid release of Ca 2ϩ is mediated by Ca 2ϩ release channels, also known as ryanodine receptors (RyRs), because they bind the plant alkaloid ryanodine with high affinity and specificity (1-6). Skeletal and cardiac muscles express two major isoforms of the RyR, RyR1 and RyR2, respectively. In striated muscles, RyRs are concentrated in the junctional SR membrane near transverse tubular, voltage-sensitive L-type Ca 2ϩ channels (dihydropyridine receptors). A muscle action potential initiates dihydropyridine receptor conformational changes that activate the RyRs via a direct physical interaction in skeletal muscle or mediate the influx of Ca 2ϩ in cardiac muscle, leading to the release of Ca 2ϩ from the SR and subsequent muscle contraction. Both RyRs have been isolated as 30 S protein complexes composed of four 560-kDa (RyR polypeptide) and four 12-kDa (FK506-binding protein) subunits (1-5). The two channel activities are affected by endogenous and exogenous effectors, such as Ca 2ϩ , Mg 2ϩ , ATP, caffeine, ryanodine, and ruthenium red.Ruthenium red (RR) is one of the most potent inhibitors of SR Ca 2ϩ release (2). RR also inhibits mitochondrial Ca 2ϩ uptake. However, this activity was due to a contaminant (7) that may be related to an oxygen-bridged R360 complex that, at concentrations as high as 10 M, was without effect on SR Ca 2ϩ uptake or release (8). RR is a polycationic dye with a linear structure consisting of three ruthenium atoms with a net valence of 6. In SR vesicles, RR increased the rate of Ca 2ϩ uptake and decreased the rate of Ca 2ϩ release at concentrations ranging from 1 nM to 20 M (9 -18). In muscle fibers, RR concentrations greater than 20 M are required to inhibit SR Ca 2ϩ release because of RR binding to myoplasmic proteins (19,20). In intact single frog twitch muscle fibers, the estimated free RR concentration for half-block of SR Ca 2ϩ release was 2.4 M, in good agreement with the range reported for SR vesicle preparations (19). In [ 3 H]ryanodine binding measurements, RR decreased the B max value and increased the K D value, with the latter effect being due to a slower association rate (16). In single channel measurements, micromolar concentrations of RR decreased the channel open probability of the skeletal (21) and cardiac (22) RyRs by producing prolonged channel closings. A different effect was observed when ryanodine was used to lock the channels into a permanently open, Ca 2ϩ -insensitive subconductance state. RR blocked single ryanodine-modified skeletal RyRs by binding in a voltage-dependent manner to multiple sites located in the conductance pore of the channel (23).The present study was undertaken to clarify the effects of RR on ryanodine-unmodified ...

Human UMP synthase is a bifunctional protein containing two separate catalytic domains, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23). These studies address the question of why the last two reactions in pyrimidine nucleotide synthesis are catalyzed by a bifunctional enzyme in mammalian cells, but by two separate enzymes in microorganisms. From existing data on subunit associations of the respective enzymes and calculations showing the molar concentration of enzyme to be far lower in mammalian cells than in microorganisms, we hypothesize that the covalent union in UMP synthase stabilizes the domains containing the respective catalytic centers. Evidence supporting this hypothesis comes from studies of stability of enzyme activity in vitro, at physiological concentrations, of UMP synthase, the two isolated catalytic domains prepared by site-directed mutagenesis of UMP synthase, and the yeast ODCase. The two engineered domains have activities very similar to the native UMP synthase, but unlike the bifunctional protein, the domains are quite unstable under conditions promoting the dissociated monomer.

The lobster skeletal muscle Ca2+ release channel, known also as the ryanodine receptor, is composed of four polypeptides of approximately 5000 amino acids each, like its mammalian counterparts. Clones encoding the carboxy-terminal region of the lobster ryanodine receptor were isolated from a lobster skeletal muscle cDNA library. Analysis of the deduced 1513 carboxy-terminal amino acid sequence suggests a cytoplasmic Ca2+ binding domain consisting of two EF-hand Ca2+ binding motifs (amino acid residues 594-656). The Ca2+ binding properties of this domain were assessed by preparing bacterial fusion proteins with sequences from the lobster Ca2+ binding domain and the corresponding sequences of the rabbit cardiac and skeletal muscle ryanodine receptors. The lobster skeletal muscle fusion protein bound 45Ca2+ in Ca2+ overlays, and bound two Ca2+ under equilibrium binding conditions with a Hill dissociation constant (KH) of 0.9 mM and coefficient (nH) of 1.4. Rabbit skeletal and cardiac fusion proteins bound two Ca2+ with KHs of 3.7 and 3.8 mM and nHs of 1.1 and 1.3, respectively. Similar to results previously reported for the mammalian RyRs, the lobster RyR was activated by micromolar Ca2+ and inhibited by millimolar Ca2+, as determined in single-channel and [3H]ryanodine binding measurements. These results suggest that the two EF-hand Ca2+ binding domain of the lobster Ca2+ release channel as well as the corresponding regions of the mammalian channels may play a role in Ca2+ inactivation of sarcoplasmic reticulum Ca2+ release.

The effects of mono- and divalent ions on Ca2+-gated cardiac muscle Ca2+-release channel (ryanodine receptor) activity were examined in [3H]ryanodine-binding measurements. Ca2+ bound with the highest apparent affinity to Ca2+activation sites in choline chloride medium, followed by KCl, CsCl, NaCl, and LiCl media. The apparent Ca2+ binding affinities of Ca2+ inactivation sites were lower in choline chloride and CsCl media than in LiCl, NaCl, and KCl media. Sr2+ activated the ryanodine receptor with a lower efficacy than Ca2+. Competition studies indicated that Li+, K+, Mg2+, and Ba2+ compete with Ca2+ for Ca2+ activation sites. In 0.125 M KCl medium, the Ca2+ dependence of [3H]ryanodine binding was modified by 5 mM Mg2+ and 5 mM β,γ-methyleneadenosine 5′-triphosphate (a nonhydrolyzable ATP analog). The addition of 5 mM glutathione was without appreciable effect. Substitution of Cl−by 2-( N-morpholino)ethanesulfonic acid ion caused an increase in the apparent Ca2+affinity of the Ca2+ inactivation sites, whereas an increase in KCl concentration had the opposite effect. These results suggest that cardiac muscle ryanodine receptor activity may be regulated by 1) competitive binding of mono- and divalent cations to Ca2+ activation sites, 2) binding of monovalent cations to Ca2+ inactivation sites, and 3) binding of anions to anion regulatory sites.