Search PubMed⌕ Search

Biomedical subjects

G Meissner

Publications and source records attributed to G Meissner.

At least 19 recordsLinked to original sources
PubMed1992

Expression of a cardiac Ca(2+)-release channel isoform in mammalian brain.

Mammalian brain possesses ryanodine-sensitive Ca2+ channels, which in muscle cells mediate rapid Ca2+ release from intracellular stores during excitation-contraction coupling. Analysis of bovine brain ryanodine receptor (RyR) channels suggests specific expression of the cardiac-muscle RyR isoform in mammalian brain. Localization using cardiac-muscle RyR-specific antibodies and antisense RNA revealed that brain RyRs were present in dendrites, cell bodies and terminals of rat forebrain, and highly enriched in the hippocampus. Activity of skeletal-muscle RyR channels is coupled to sarcolemmal voltage sensors, in contrast with cardiac-muscle RyR channels, which are known to be Ca(2+)-induced Ca(2+)-release channels. Thus Ca(2+)-induced Ca2+ release from intracellular stores mediated by brain RyR channels may be a major Ca(2+)-signalling pathway in specific regions of mammalian brain, and hence may play a fundamental role in neuronal Ca2+ homoeostasis.

Animals↗
PubMed1992

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel.

Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca2+ release channel (ryanodine receptor) in excitable tissues. Using [3H]ryanodine, a 30 S protein complex comprised of four polypeptides of Mr 565,000 has been isolated and functionally reconstituted into planar lipid bilayers. The effects of salt concentration and divalent cations on skeletal muscle sarcoplasmic reticulum [3H]ryanodine binding and Ca2+ release channel activity have been compared. These studies suggest that ryanodine is a good probe for investigating the function of the release channel.

Animals↗
PubMed1992

The 30 S lobster skeletal muscle Ca2+ release channel (ryanodine receptor) has functional properties distinct from the mammalian channel proteins.

The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized ryanodine receptor (RyR) of lobster skeletal muscle has been isolated by rate density centrifugation as a 30 S protein complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified 30 S receptor revealed a single high molecular weight protein band with a mobility intermediate between those of the mammalian skeletal and cardiac M(r) 565,000 RyR polypeptides. Immunoblot analysis showed no or only minimal cross-reactivity with the rabbit skeletal and canine cardiac RyR polypeptides. By immunofluorescence the lobster RyR was localized to the junctions of the A-I bands. Following planar lipid bilayer reconstitution of the purified 30 S lobster RyR, single channel K+ and Ca2+ currents were observed which were modified by ryanodine and optimally activated by millimolar concentrations of cis (cytoplasmic) Ca2+. Vesicle-45Ca2+ flux measurements also indicated an optimal activation of the lobster Ca2+ channel by millimolar Ca2+, whereas 45Ca2+ efflux from mammalian skeletal and cardiac muscle sarcoplasmic reticulum (SR) vesicles is optimally activated by micromolar Ca2+. Further, mammalian muscle SR Ca2+ release activity is modulated by Mg2+ and ATP, whereas neither ligand appreciably affected 45Ca2+ efflux from lobster SR vesicles. These results suggested that lobster and mammalian muscle express immunologically and functionally distinct SR Ca2+ release channel protein complexes.

Animals↗
PubMed1992

Effects of regional ischemia on the ryanodine-sensitive Ca2+ release channel of canine cardiac sarcoplasmic reticulum.

In mammalian myocardium, muscle contraction is regulated by the rapid release of Ca2+ ions through ryanodine-sensitive Ca2+ release channels present in the intracellular membrane compartment, sarcoplasmic reticulum (SR). In this study, the effects of regional ischemia on intrinsic SR Ca2+ release channel function were determined by studying the Ca2+ transport and release, and [3H]ryanodine binding properties of whole muscle homogenates and SR-enriched membrane fractions from normal and ischemic myocardium. Measurement of oxalate-supported 45Ca(2+)-uptake rates before and after pretreatment with 1 mM ryanodine, indicated that the SR Ca2+ release channel retained its ability to be effectively closed by the channel-specific probe ryanodine after 15 and 60 min of ischemia. 45Ca2+ efflux from, and high-affinity [3H]ryanodine binding to SR-enriched vesicle fractions indicated retention of regulation of Ca2+ release channel activity by Ca2+, Mg2+ and adenine nucleotide in 15 and 60 min ischemic samples. Further, sodium dodecylsulfate polyacrylamide gel and immunoblot analysis revealed no proteolytic degradation of the M(r) 565,000 SR Ca2+ release channel polypeptide after 15 and 60 min of ischemia. These results suggested a minimal, if any, loss of intrinsic SR Ca2+ release channel function in ischemic hearts.

Animals↗
PubMed1992

Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle.

The ryanodine receptor (RyR)-Ca2+ release channels of frog skeletal muscle have been purified as 30S protein complexes comprised of two high molecular weight polypeptides. The upper and lower bands of the frog doublet comigrated on sodium dodecyl sulfate polyacylamide gels with the mammalian skeletal and cardiac RyR polypeptides, respectively. Immunoblot analysis showed that a polyclonal antiserum to the rat skeletal RyR preferentially cross-reacted with the upper band, whereas monoclonal antibodies to the canine cardiac RyR preferentially cross-reacted with the lower band of the frog receptor doublet. Immunoprecipitation studies indicated the presence of two homooligomer 30S RyR complexes comprised of either the lower or upper polypeptide band of the frog doublet, and immunocytochemical staining revealed their colocalization in frog gastrocnemius muscle. After planar lipid bilayer reconstitution of the 30S frog RyR, single-channel currents were observed that exhibited a Na+ and Ca2+ conductance and pharmacological characteristics similar to those of the mammalian skeletal and cardiac Ca2+ release channels. These results suggest that amphibian skeletal muscle expresses two distinct RyR isoforms that share epitopes in common with the mammalian skeletal or cardiac RyR.

Animals↗
PubMed1991

Reconstitution of the solubilized cardiac sarcoplasmic reticulum potassium channel. Identification of a putative Mr approximately 80 kDa polypeptide constituent.

Recent evidence has indicated that potassium ion movement through sarcoplasmic reticulum (SR) K+ channels is an important countercurrent for Ca2+ release from SR. We used Chaps-solubilized SR vesicles and sucrose density gradient centrifugation to identify components of the canine cardiac SR K+ channel. To overcome the difficulty of the absence of a high-affinity specific ligand, we have successfully applied the planar lipid bilayer reconstitution technique to identify and functionally assay for the solubilized SR K+ channel. We found that Chaps solubilization of the channel did not change the protein's functional properties. The cardiac SR K+ channel sediments as a 15-20S protein complex. A polypeptide of Mr approximately 80 kDa was found to specifically comigrate with the 15-20S gradient fractions and might be a major constituent of the cardiac SR K+ channel.

Animals↗
PubMed1991

Functional characterization of the Ca(2+)-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum.

The Ca(2+)-gated Ca2+ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulphonate (CHAPS) in the presence and absence of 3[H]-ryanodine and centrifuged through linear sucrose gradients. A single 3[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca(2+)- and monovalent-ion-conducting channel (110 pS in 100 mM Ca2+, 360 pS in 250 mM K+). The channel was activated by micromolar Ca2+, modulated by millimolar adenosine triphosphate, Mg2+ and the Ca(2+)-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca(2+)-gated Ca2+ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca2+ release channel complexes.

Animals↗
PubMed1990

Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum.

We have cloned cDNAs encoding the rabbit and human forms of the Ca2+ release channel of sarcoplasmic reticulum. The human cDNA encodes a protein of 5032 amino acids, with a molecular weight of 563,584, which is made without an NH2-terminal signal sequence. Amino acid substitutions between rabbit and human sequences were noted in 163 positions and deletions or insertions in eight regions accounted for additional sequence differences between the two proteins. Analysis of the sequence indicates that 10 potential transmembrane sequences in the COOH-terminal fifth of the molecule and two additional, potential transmembrane sequences nearer to the center of the molecule could contribute to the formation of the Ca2+ conducting pore. The remainder of the molecule is hydrophilic and presumably constitutes the cytoplasmic domain of the protein. A 114-120 amino acid motif is repeated four times in the protein, in residues 841-954, 955-1068, 2725-2844, and 2845-2958 and a 16 amino acid part of the motif is repeated twice more in residues 1344-1359 and 1371-1386. Although the channel is modulated by Ca2+, ATP, and calmodulin, no clear high affinity Ca2(+)-binding domain of the EF hand type and no clear high affinity ATP-binding domain were detected in the primary sequence. An acidic sequence in residues 1872-1923 contains 79% glutamate or aspartate residues and this sequence is a potential low affinity Ca2(+)-binding site. Several potential calmodulin-binding sites were observed in the sequence, in the region 2800 to 3050.

Amino Acid Sequence↗
PubMed1989

Activation of the skeletal muscle Ca2+ release channel by the triazine dyes cibacron blue F3A-G and reactive red 120.

Vesicle-45Ca2+ ion flux and planar lipid bilayer single-channel measurements have shown that the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (SR) is activated by micromolar concentrations of Cibacron Blue F3A-G (Reactive Blue 2) and Reactive Red 120. Cibacron Blue increased the 45Ca2+ efflux rate from heavy SR vesicles by apparently interacting with both the adenine nucleotide and caffeine activating sites of the channel. Dye-induced 45Ca2+ release was inhibited by Mg2+ and ruthenium red. In single channel recordings with the purified channel protein complex, Cibacron Blue increased the open time of the Ca2+ release channel without an apparent change in the conductance of the main and subconductance states of the channel.

Adenine↗
PubMed1989

The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. Evidence for a cooperatively coupled, negatively charged homotetramer.

The subunit structure of the rabbit skeletal muscle ryanodine receptor-Ca2+ release channel complex was examined following solubilization of heavy sarcoplasmic reticulum membranes in two zwitterionic detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Zwittergent 3-14. High and low affinity [3H]ryanodine binding was retained upon solubilization of the complex in Chaps but was lost in Zwittergent 3-14. The purified complex migrated as a single peak with an apparent sedimentation coefficient of approximately 30 and approximately 9 S upon density gradient centrifugation and with isoelectric points of 3.7 and 3.9 upon two-dimensional gel electrophoresis in Chaps and Zwittergent 3-14, respectively. Electron microscopy of negatively stained samples indicated that the distinct four-leaf clover structure of the ryanodine receptor observed in Chaps disappeared following Zwittergent treatment of the 30 S complex and instead showed smaller, round particles. Ferguson plot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial and fully cross-linked and incompletely denatured complexes suggested a stoichiometry of four Mr approximately 400,000 peptides/30 S ryanodine receptor oligomer. [3H]Ryanodine binding to the membrane-bound receptor in 50 microM--1 mM free Ca2+ revealed the presence of both high affinity (KD = 8 nM, Hill coefficient (nH) = 0.9) and low affinity (nH approximately 0.45) sites with a ratio of 1:3. Reduction in free Ca2+ to less than or equal to 0.1 microM or trypsin digestion of the membranes resulted in loss of high affinity but not low affinity ryanodine binding (Hill KD = 5,000 nM, nH = 0.9). Addition of 20 mM caffeine to the nanomolar Ca2+ medium decreased the Hill KD to 1,000 nM without changing the Hill coefficient. Occupation of the low affinity sites altered the rate of [3H]ryanodine dissociation from the high affinity sites. Single channel recordings of the purified ryanodine receptor channel incorporated into planar lipid bilayers also indicated the existence of high and low affinity sites for ryanodine, occupation of which resulted in formation of a subconducting and completely closed state of the channel, respectively. These results are compatible with a subunit structural model of the 30 S ryanodine receptor-Ca2+ release channel complex which comprises a homotetramer of negatively charged and allosterically coupled polypeptides of Mr approximately 400,000.

Animals↗
PubMed1989

Structural and functional correlation of the trypsin-digested Ca2+ release channel of skeletal muscle sarcoplasmic reticulum.

The effect of trypsin digestion on the (i) fragmentation pattern, (ii) activity, (iii) [3H]ryanodine binding, and (iv) sedimentation behavior of the skeletal sarcoplasmic reticulum (SR) ryanodine receptor-Ca2+ release channel complex has been examined. Mild tryptic digestion of heavy, junctional-derived SR vesicles resulted in the rapid disappearance of the high molecular weight (Mr approximately 400,000) Ca2+ release channel protein on sodium dodecyl sulfate gels and appearance of bands of lower Mr upon immunoblot analysis, without an appreciable effect on [3H]ryanodine binding or the apparent S value (30 S) of the 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized channel complex. Further degradation to bands of Mr greater than 70,000 on immunoblots correlated with a reduction of channel size from 30 S to 10-15 S and loss of high affinity [3H]ryanodine binding to the trypsinized receptor, while low affinity [3H]ryanodine binding and [3H]ryanodine bound prior to digestion were retained. Parallel 45Ca2+ efflux measurements also indicated retention of the Ca2+, Mg2+, and ATP regulatory sites, although Ca2+-induced 45Ca2+ release rates were changed. In planar lipid bilayer-single channel measurements, addition of trypsin to the cytoplasmic side of the high conductance (100 pS in 50 mM Ca2+), Ca2+-activated SR Ca2+ channel initially increased the fraction of channel open time and was followed by a complete and irreversible loss of channel activity. Trypsin did not change the unitary conductance, and was without effect on single channel activity when added to the lumenal side of the channel.

Adenosine Monophosphate↗
PubMed1989

Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex.

Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle.

Animals↗
PubMed1989

The muscle ryanodine receptor and its intrinsic Ca2+ channel activity.

In skeletal and cardiac muscle, contraction is initiated by the rapid release of Ca2+ ions from the intracellular membrane system, sarcoplasmic reticulum. Rapid-mixing vesicle ion flux and planar lipid bilayer-single-channel measurements have shown that Ca2+ release is mediated by a high-conductance, ligand-gated Ca2+ channel. Using the Ca2+ release-specific probe ryanodine, a 30 S protein complex composed of four polypeptides of Mr approximately 400,000 has been isolated. Reconstitution of the purified skeletal and cardiac muscle 30 S complexes into planar lipid bilayers induced single Ca2+ channel currents with conductance and gating kinetics similar to those of native Ca2+ release channels. Electron microscopy revealed structural similarity with the protein bridges ("feet") that span the transverse-tubule-sarcoplasmic reticulum junction. These results suggest that striated muscle contains an intracellular Ca2+ release channel that is identical with the ryanodine receptor and the transverse-tubule-sarcoplasmic reticulum spanning feet structures.

Animals↗
PubMed1989

Isolation of a Ca2(+)-releasing factor from caffeine-treated skeletal muscle fibres and its effect on Ca2+ release from sarcoplasmic reticulum.

In the presence of 2 mM caffeine, skeletal muscles of the frog exert small irregular oscillations of single sarcomeres. A factor, released from these oscillating muscles, was partially purified, and its activity was tested on skinned fibres and isolated vesicles of the sarcoplasmic reticulum (SR). Purification was achieved in three steps by gel filtration and reversed phase chromatography, and the active compound of the released material was shown probably to be a small peptide. In skinned fibres, the purified factor evoked repetitive contractions and subthreshold sarcomeric oscillations. In 'heavy' SR vesicles passively loaded with 45Ca2+, the factor induced a small but significant increase in the 45Ca2+ efflux rate. At the single channel level, the open probability of the SR Ca2+ release channel increased when the factor was added to the cytoplasmic side of the channel. The results reveal that the released factor potentiates Ca2+ release from the SR by increasing the open time of the Ca2+ release channel.

Amino Acids↗