Biomedical subjects

V Shoshan-Barmatz

Publications and source records attributed to V Shoshan-Barmatz.

At least 19 recordsLinked to original sources

PubMed2006

The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death.

Research over the last decade has extended the prevailing view of mitochondria to include functions well beyond the critical bioenergetics role in supplying ATP. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organelle communication, aging, many diseases, cell proliferation and cell death. Apoptotic signal transmission to the mitochondria results in the efflux of a number of potential apoptotic regulators to the cytosol that trigger caspase activation and lead to cell destruction. Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) is involved in this release of proteins via the outer mitochondrial membrane. VDAC in the outer mitochondrial membrane is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. VDAC has been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and anti-apoptotic Bcl2-family of proteins and due to its function in the release of apoptotic proteins located in the inter-membranal space. The diameter of the VDAC pore is only about 2.6-3 nm, which is insufficient for passage of a folded protein like cytochrome c. New work suggests pore formation by homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins such as Bax or Bak. This review provides insights into the central role of VDAC in cell life and death and emphasizes its function in the regulation of mitochondria-mediated apoptosis and, thereby, its potential as a rational target for new therapeutics.

Animals↗

PubMed2005

The voltage-dependent anion channel in endoplasmic/sarcoplasmic reticulum: characterization, modulation and possible function.

In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca(2+) homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca(2+)and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca(2+) homeostasis and mitochondria-mediated apoptosis will be discussed.

Animals↗

PubMed2005

The voltage-dependent anion channel-1 modulates apoptotic cell death.

The role of the voltage-dependent anion channel (VDAC) in cell death was investigated using the expression of native and mutated murine VDAC1 in U-937 cells and VDAC inhibitors. Glutamate 72 in VDAC1, shown previously to bind dicyclohexylcarbodiimide (DCCD), which inhibits hexokinase isoform I (HK-I) binding to mitochondria, was mutated to glutamine. Binding of HK-I to mitochondria expressing E72Q-mVDAC1, as compared to native VDAC1, was decreased by approximately 70% and rendered insensitive to DCCD. HK-I and ruthenium red (RuR) reduced the VDAC1 conductance but not that of E72Q-mVDAC1. Overexpression of native or E72Q-mVDAC1 in U-937 cells induced apoptotic cell death (80%). RuR or overexpression of HK-I prevented this apoptosis in cells expressing native but not E72Q-mVDAC1. Thus, a single amino-acid mutation in VDAC prevented HK-I- or RuR-mediated protection against apoptosis, suggesting the direct VDAC regulation of the mitochondria-mediated apoptotic pathway and that the protective effects of RuR and HK-I rely on their binding to VDAC.

Amino Acid Substitution↗

PubMed2001

Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function.

Mitochondria play a central role in energy metabolism, Ca(2+) signalling, aging and cell death. To control cytosolic or mitochondrial Ca(2+) concentration, mitochondria possess several Ca(2+)-transport systems across the inner membrane. However, the pathway for Ca(2+) crossing the outer membrane has not been directly addressed. We report that purified voltage-dependent anion channel (VDAC) reconstituted into lipid bilayers or liposomes is highly permeable to Ca(2+). VDAC contains Ca(2+)-binding sites that bind Ruthenium Red (RuR), La(3+) and that RuR completely closed VDACs in single or multichannel experiments. Energized, freshly prepared mitochondria accumulate Ca(2+) (500-700 nmol/mg of protein), and subsequently released it. The release of Ca(2+) is accompanied by cyclosporin A-inhibited swelling, suggesting activation of permeability transition pore (PTP). RuR and ruthenium amine binuclear complex, when added to mitochondria after Ca(2+) accumulation has reached a maximal level and before PTP is activated, prevented the release of Ca(2+) and the accompanied mitochondrial swelling. RuR also prevented PTP opening promoted by atractyloside, an adenine nucleotide translocase inhibitor. These results suggest that VDAC, located in the mitochondrial outer membrane, controls Ca(2+) transport into and from the mitochondria, and that the inhibition of Ca(2+) uptake by RuR and La(3+) may result from their interaction with VDAC Ca(2+)-binding sites. Inhibition of PTP opening or assembly by RuR and ruthenium amine binuclear complex suggest the involvement of VDAC in PTP activity and/or regulation. The permeability of VDAC to Ca(2+) and its binding of Ca(2+), suggest that VDAC has a role in regulation of the mitochondrial Ca(2+) homoeostasis.

Animals↗

PubMed2000

Modulation of the voltage-dependent anion channel (VDAC) by glutamate.

The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is a large channel permeable to anions, cations, ATP, and other metabolites. VDAC was purified from sheep brain synaptosomes or rat liver mitochondria using a reactive red-agarose column, in addition to the hydroxyapatitate column. The red-agarose column allowed further purification (over 98%), concentration of the protein over ten-fold, decreasing Triton X-100 concentration, and/or replacing Triton X-100 with other detergents, such as Nonidet P-40 or octylglucoside. This purified VDAC reconstituted into planar-lipid bilayer, had a unitary maximal conductance of 3.7 +/- 0.1 nS in 1 M NaCl, at 10 mV and was permeable to both large cations and anions. In the maximal conducting state, the permeability ratios for Na(+), acetylcholine(+), dopamine,(+) and glutamate(-), relative to Cl(-), were estimated to be 0.73, 0.6, 0.44, and 0.4, respectively. In contrast, in the subconducting state, glutamate(-) was impermeable, while the relative permeability to acetylcholine(+) increased and to dopamine(+) remained unchanged. At the high concentrations (0.1-0.5 M) used in the permeability experiments, glutamate eliminated the bell shape of the voltage dependence of VDAC channel conductance. Glutamate at concentrations of 1 to 20 mM, in the presence of 1 M NaCl, was found to modulate the VDAC channel activity. In single-channel experiments, at low voltages (+/-10 mV), glutamate induced rapid fluctuations of the channel between the fully open state and long-lived low-conducting states or short-lived closed state. Glutamate modification of the channel activity, at low voltages, is dependent on voltage, requiring short-time (20-60 sec) exposure of the channel to high membrane potentials. The effect of glutamate is specific, since it was observed in the presence of 1 M NaCl and it was not obtained with aspartate or GABA. These results suggest that VDAC possesses a specific glutamate-binding site that modulates its activity.

Acetylcholine↗

PubMed1999

Modification of ryanodine receptor/Ca2+ release channel with dinitrofluorobenzene.

Modification of the ryanodine receptor (RyR)/Ca(2+) release channel with 2,4-dinitrofluorobenzene (DNFB) indicated that two classes of amino group interact with the reagent, as can be distinguished on the basis of their reactivity/accessibility and the effects on ryanodine binding and single channel activities. One group interacted very rapidly (t(1/2)<30 s) at 25 degrees C with low concentrations of DNFB [C(50) (concentration of DNFB required for 50% inhibition or stimulation of ryanodine binding)=5 microM], and at pH values of 6.2 and higher. This interaction resulted in the marked stimulation of ryanodine binding and the complete inhibition of a single Ca(2+) release channel incorporated into planar lipid bilayer. The second group is accessible at higher temperatures (37 degrees C); at pH values higher than 7.4 it reacted slowly (t(1/2)=20 min) with high concentrations of DNFB (C(50)=70 microM). This interaction led to the inhibition of ryanodine binding and single channel activity. Modification of RyR with DNFB under the stimulatory conditions resulted in 3.6-fold and 6-fold increases in ryanodine-binding and Ca(2+)-binding affinities respectively. Modification with DNFB under the inhibitory conditions resulted in a decrease in the total ryanodine-binding sites. The exposure of the RyR single channel to DNFB under both inhibitory and stimulatory conditions led to the complete closure of the channel. However, when modified under the stimulatory conditions, but not under the inhibitory ones, the DNFB-modified closed channel could be re-activated by sub-micromolar concentrations of ryanodine, in the presence of nanomolar concentrations of Ca(2+). The DNFB-modified ryanodine-activated RyR channel showed fast transitions between open, closed and several sub-conductance states, and was completely closed by Ruthenium Red. ATP re-activated the DNFB-modified closed channel or, if present during modification, prevented the inhibition of RyR channel activity by DNFB. Neither the stimulation nor the inhibition of ryanodine binding by modification with DNFB was affected by the presence of ATP. By using the photoreactive ATP analogue 3'-O-(4-benzoyl)benzoyl-[alpha-(32)P]ATP we found that DNFB modification had no effect on the ATP-binding site of RyR. The results are discussed with regard to the involvement of amino group residues in channel gating, ryanodine association/dissociation and occlusion, and the relationship between the open/closed state of the RyR and its capacity to bind ryanodine.

Adenosine Triphosphate↗

PubMed1999

Characterization of sheep brain ryanodine receptor ATP binding site by photoaffinity labeling.

Two high Mr protein bands (440 and 420 kDa) in sheep brain microsomal membranes were labeled with the photoaffinity ATP analog, O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP). The 420 kDa band is labeled by [alpha-32P]-Bz2ATP with about 1000-fold higher affinity than the 440 kDa band. The heavily labeled 420 kDa band is identified as dynein heavy chain based on its partial amino acid sequence, and cross-reactivity with anti-dynein antibodies. The 440 kDa protein is immunologically identified as the type-2 RyR. Bz2ATP binding is obtained in the absence of divalent cations. Bz2ATP and ATP increased the binding of ryanodine to its receptor up to 3-fold, and increased the binding affinity up to 6-fold. Other nucleotides stimulate ryanodine binding with decreasing effectiveness: Bz2ATP > ATP > ADP > AMP > AMP-PNP > GTP > cAMP. With respect to nucleotide specificity, this binding site is similar to the skeletal muscle RyR (type 1). However, the brain RyR may have additional one or more sites with lower affinity with inhibitory effect on ryanodine binding. These results suggest that the major RyR isoform in sheep brain corresponds to the type-2 isoform, and that modulation of ryanodine binding by ATP involves its binding to the RyR protein. The association of dynein with brain microsomal membranes may reflect a linkage of RyR to the cytoskeleton.

Adenosine Triphosphate↗

PubMed1999

Cardiac sarcalumenin: phosphorylation, comparison with the skeletal muscle sarcalumenin and modulation of ryanodine receptor.

Cardiac sarcoplasmic reticulum (SR) contains an endogenous phosphorylation system that under specific conditions phosphorylates two proteins with apparent molecular masses of 150 and 130 kDa. The conditions for their phosphorylation are as for the skeletal muscle sarcalumenin and the histidine-rich Ca2+ binding protein (HCP) with respect to: (i) Ca2+ and high concentrations of NaF are required; (ii) phosphorylation is obtained with no added Mg2+ and shows a similar time course and ATP concentration dependence; (iii) inhibition by similar concentrations of La3+; (iv) phosphorylation is obtained with [gamma-32P]GTP; (v) ryanodine binding is inhibited parallel to the phosphorylation of the two proteins. The endogenous kinase is identified as casein kinase II (CK II) based on its ability to use GTP as effectively as ATP, and its inhibition by La3+. The association of CK II with the cardiac SR, even after EGTA extraction at alkaline pH, is demonstrated using antibodies against CK II. The cardiac 130 kDa protein is identified as sarcalumenin based on its partial amino acid sequence and its blue staining with Stains-All. Cardiac sarcalumenin is different from the skeletal muscle protein based on electrophoretic mobilities, immunological analysis, peptide and phosphopeptide maps, as well as amino acid sequencing. Preincubation of SR with NaF and ATP, but not with NaF and AMP-PNP caused strong inhibition of ryanodine binding. This is due to decrease in Ca2+- and ryanodine-binding affinities of the ryanodine receptor (RyR) by about 6.6 and 18-fold, respectively. These results suggest that cardiac sarcalumenin is an isoform of the skeletal muscle protein. An endogenous CK II can phosphorylate sarcalumenin, and in parallel to its phosphorylation the properties of the ryanodine receptor are modified.

Adenosine Triphosphate↗

PubMed1998

Dicyclohexylcarbodiimide interaction with the voltage-dependent anion channel from sarcoplasmic reticulum.

In a recent study [Shoshan-Barmatz, V., Orr, I., Weil, S., Meyer, H., Varsanyi, M. & Heilmeyer, L. M. G. (1996) FEBS Lett. 386, 205-210] we have demonstrated the presence of the voltage-dependent anion channel (VDAC) in skeletal muscle sarcoplasmic reticulum (SR) as supported here by co-localization of VDAC and (Ca2+ + Mg2+)ATPase in the SR using double-immunogold labeling. The interaction of the carboxyl-modifying reagent dicyclohexylcarbodiimide with the SR-VDAC is characterized by labeling with [14C]dicyclohexylcarbodiimide and by dicyclohexylcarbodiimide modification of the reconstituted-purified VDAC channel activity. In both SR and mitochondrial membranes, [14C]dicyclohexylcarbodiimide most specifically labeled a 35-kDa protein, identified as VDAC by specific anti-VDAC Ig. Labeling of the SR-VDAC was about twofold higher than that of the mitochondrial VDAC, which could result either form higher labeling of the SR protein or from relatively higher amounts of VDAC/mg total protein in the SR membranes. [14C]Dicyclohexylcarbodiimide labeling of the SR, but not the mitochondrial VDAC, was biphasic with respect to time and concentration of [14C]dicyclohexylcarbodiimide. Partial digestion of [14C]dicyclohexylcarbodiimide-labeled SR-VDAC with chymotrypsin yielded five proteolytic fragments which were recognized by the anti-VDAC Ig, and the dicyclohexylcarbodiimide-binding site was localized in the 19-kDa fragment. VDAC was purified from SR and mitochondrial membranes by spermine-agarose column. The interaction of dicyclohexylcarbodiimide with functional carboxyl residue(s) in the purified VDAC is demonstrated by recording its channel activity, following its reconstitution into planar lipid bilayer (PLB). Dicyclohexylcarbodiimide inhibited the channel activity in a voltage-dependent manner, requiring incubation with dicyclohexylcarbodiimide at high (negative or positive) potentials. Dicyclohexylcarbodiimide slowed down the transition from the high-conducting to a long-lived low-conducting states of the channel (approximately 20% of its maximal conductance), by stabilizing the intermediate states. Similar results were also obtained with purified-reconstituted mitochondrial VDAC. Hydrophilic carboxyl reagents [[1-ethyl-3-(3-dimethylamino)propyl] carbodiimide, N-ethyl-phenylisoxazolium-3'-sulfonate] neither modified the channel activity nor prevented [14C]dicyclohexylcarbodiimide labeling. These results indicate that dicyclohexylcarbodiimide interacts with a carboxyl group located in a hydrophobic region of the protein which is involved in the channel gating.

Animals↗

PubMed1998

Identification, characterization and partial purification of a thiol-protease which cleaves specifically the skeletal muscle ryanodine receptor/Ca2+ release channel.

A 94 kDa large subunit thiol-protease, as identified by anti-calpain antibodies, has been isolated from skeletal muscle junctional sarcoplasmic reticulum (SR). This protease cleaves specifically the skeletal muscle ryanodine receptor (RyR)/Ca2+ release channel at one site resulting in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease degrades neither other SR proteins nor the ryanodine receptor of cardiac nor brain membranes. The partially purified 94 kDa protease, like the SR associated protease, had an optimal pH of about 7.0, was absolutely dependent on the presence of thiol reducing reagents, and was completely inhibited by HgCl2, leupeptin and the specific calpain I inhibitor. However, while the SR membrane-associated protease requires Ca2+ at a submicromolar concentration, the isolated thiol-protease has lost the Ca2+ requirement. The 94 kDa thiol-protease had no effect on ryanodine binding but modified the channel activity of RyR reconstituted into planar lipid bilayer: in a time-dependent manner, the channel activity decreases and within several minutes the channel is converted into a subconducting state. The protease-modified channel activity is still Ca2+-dependent and ryanodine sensitive. This 94 kDa thiol-protease cross react with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal muscle specific calpain p94.

Animals↗

PubMed1998

The structure, function, and cellular regulation of ryanodine-sensitive Ca2+ release channels.

The fundamental biological process of Ca2+ signaling is known to be important in most eukaryotic cells, and inositol 1,2,5-trisphosphate and ryanodine receptors, intracellular Ca2+ release channels encoded by two distantly related gene families, are central to this phenomenon. Ryanodine receptors in the sarcoplasmic reticulum of skeletal and cardiac muscle have a predominant role in excitation-contraction coupling, but the channels are also present in the endoplasmic reticulum of noncontractile tissues including the central nervous system and the immune system. In all, three highly homologous ryanodine receptor isoforms have been identified, all very large proteins which assemble as (homo)tetramers of approximately 2 MDa. They contain large cytoplasmically disposed regulatory domains and are always associated with other structural or regulatory proteins, including calmodulin and immunophilins, which can have marked effects on channel function. The type 1 isoform in skeletal muscle is electromechanically coupled to surface membrane voltage sensors, whereas the remaining isoforms appear to be activated solely by endogenous cytoplasmic second messengers or other ligands, including Ca2+ itself ("Ca(2+)-induced Ca2+ release"). This review concentrates on ryanodine receptor structure-function relationships as probed by a variety of methods and on the molecular mechanisms of channel modulation at the cellular level (including evidence for the regulation of gene expression and transcription). It also touches on the relevance of ryanodine receptors to complex cellular functions and disease.

Animals↗

PubMed1997

Involvement of lysine residues in the gating of the ryanodine receptor/Ca2+-release channel of skeletal muscle sarcoplasmic reticulum.

In this study, the modification of skeletal muscle ryanodine receptor (RyR)/Ca2+-release channel with 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole (Nbd-Cl) demonstrates that lysyl residues are involved in the channel gating. Nbd-Cl was found to have a dual effect: stimulation and inhibition of ryanodine binding and single channel activities. Nbd-Cl, in a time-dependent manner, first stimulated and subsequently inhibited ryanodine binding to both membrane-bound and purified RyR. Incubation of sacroplasmic reticulum membranes with Nbd-Cl for 5-20 s resulted in enhanced ryanodine-binding activity by 2-4-fold due, to an increased binding affinity by about tenfold, with no effect on the total binding sites (Bmax). However, under prolonged incubation (5-20 min), Nbd-Cl strongly inhibited ryanodine binding by decreasing the Bmax with no effect on the binding affinity. Similar effects of stimulation and inhibition by Nbd-Cl were obtained with single channel activity of RyR reconstituted into planar lipid bilayer. Nbd-Cl initially (within a few seconds) activated the channel to a highly open state, then (within a few minutes) inactivated it to the completely closed state. Nbd-Cl-modified protein, as assayed by ryanodine binding or single channel activities, was stable against thiolysis by dithiothreitol, suggesting Nbd-Cl modification of lysyl residues. Evidence from absorption and fluorescence excitation and emission spectra also demonstrated that lysyl residues in RyR were modified by Nbd-Cl. Spectrophotometric data were used to estimate a ratio of up to 1 mol Nbd bound/mol RyR (tetramer) and up to 4 mol Nbd bound per mol RyR (tetramer) for Nbd-Cl stimulated and inhibited RyR activities, respectively. The results clearly indicate the involvement of two classes of lysyl residues in RyR activity. Modification by Nbd-Cl of the fast-reacting group led to stimulation of ryanodine binding and single channel activities, while modification of the slow-reacting group resulted in inhibition of these activities. Thus, the involvement of lysine residues in the gating of the RyR channel is proposed.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

Modulation of the skeletal muscle ryanodine receptor by endogenous phosphorylation of 160/150-kDa proteins of the sarcoplasmic reticulum.

This paper demonstrates and characterizes the inhibition of ryanodine binding caused by the phosphorylation of the 160/150-kDa proteins in skeletal muscle sarcoplasmic reticulum (SR). Inhibition of ryanodine binding was obtained by preincubation of SR membranes with ATP + NaF . The inhibition was characterized by the following findings: (a) If ATP was replaced by AdoPP[NH]P, inhibition of ryanodine binding activity was not observed. (b) The inhibitory effect of preincubation with ATP + NaF, like the phosphorylation of 150/160-kDa proteins, was Ca2+ dependent. (c) Inhibition of ryanodine binding, as the protein phosphorylation, was not observed if NaF (> 30 mM) was replaced with okadaic acid. (d) The optimal pH for the inhibition and the phosphorylation was about 7.0. (e) Both the phosphorylation of the 160/150-kDa proteins and inhibition of ryanodine binding were prevented by dichlorobenzimidazole riboside and hemin, inhibitors of casein kinase II. (f) Dephosphorylation of the 160/150-kDa proteins prevented the inhibition of ryanodine binding. (g) The presence of NP-40 during the phosphorylation prevented both the 160/150-kDa phosphorylation and the inhibition of ryanodine binding. Furthermore, a linear relationship was obtained between the degree of ryanodine binding inhibition and the level of phosphorylation of the 160/150-kDa proteins, as controlled by ATP or NaF concentrations. The binding affinity for Ca2+ of the ryanodine receptor (RyR) was modified by phosphorylation of the 160/150-kDa proteins, decreasing by up to 100-fold. The phosphorylation of the SR membranes resulted in an elimination of ryanodine binding sites with slight effect on the ryanodine binding affinity. These results suggest the modulation of the properties of the RyR by phosphorylation/dephosphorylation of the 160/150-kDa proteins. The identification of the phosphorylated 160/150-kDa proteins, their kinase, and the structural interactions between them and the RyR are presented in the accompanying paper.

Adenosine Triphosphate↗

PubMed1996

The identification of the phosphorylated 150/160-kDa proteins of sarcoplasmic reticulum, their kinase and their association with the ryanodine receptor.

In the present work we studied the relationship between the phosphorylated 150- and 160-kDa proteins and other SR proteins in the 150,000-170,000 range of molecular masses. on SDS-PAGE, the identification of their kinase, as well as the purification and structural interactions between these proteins and the rynodine receptor (RyR). The phosphorylated 150-kDa protein was identified as sarcalumenin based on: (a) its cross-reactivity with three different monoclonal antibodies specific for sarcalumenin. (b) its mobility in SDS-PAGE which was modified upon digestion with endoglycosidase H, (c) its elution from lentil-lectin column by alpha-methyl mannoside, (d) its resistance to trypsin, (e) its ability to bind Ca2+ and to stain blue with Stains-All. The phosphorylated 160-kDa protein was identified as the histidine-rich Ca2+ binding protein (HCP) based on: (a) its Ca(2+)-binding property and staining blue with Stains-All, (b) phosphorylation with the catalytic subunit of cAMP-dependent kinase. (c) its increased mobility in SDS-PAGE in the presence of Ca2+ (d) its heat stability and (e) its partial amino acid sequence. The endogenous kinase was identified as casein kinase II (CK II) based on the inhibition of the endogenous phosphorylation 160/150-kDa proteins by heparin, 5.6-dichlorobenzimidazole riboside, polyaspartyl peptide and hemin, and its ability to use [gamma-32P]GTP as the phosphate donor. The association of CK II with SR membranes, was demonstrated using specific polyclonal anti-CK II antibodies. The luminal location of CK II is suggested because CK II was extracted from the SR by l M NaCl only after their treatment with hypotonic medium, and CK II activity was inhibited with the charged inhibitors heparin and polyaspartyl peptide only after their incubation with the SR in the presence of NP-40. The 160- and 150-kDa proteins were purified on spermine-agarose column, and were phosphorylated by CK II. Like the endogenous phosphorylation of the 150/160-kDa proteins in SR. the phosphorylation of the purified proteins by CK II was inhibited by La3+ (Cl50 = 4 microM) and hemin. The results suggest the phosphorylation of the luminally located sarcalumenin and HCP with CK II.

Animals↗

PubMed1996

VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle.

In this study we demonstrate the existence of a protein with properties of the voltage-dependent anion channel (VDAC) in the sarcoplasmic reticulum (SR) using multiple approaches as summarized in the following: (a) 35 and 30 kDa proteins in different SR preparations, purified from other membranal systems by Ca2+/oxalate loading and sedimentation through 55% sucrose, cross-react with four different VDAC monoclonal antibodies. (b) Amino acid sequences of three peptides derived from the SR 35 kDa protein are identical to the sequences present in VDAC1 isoform. (c) Similar to the mitochondrial VDAC, the SR protein is specifically labeled by [14C]DCCD. (d) Using a new method, a 35 kDa protein has been purified from SR and mitochondria with a higher yield for the SR. (e) Upon reconstitution into a planar lipid bilayer, the purified SR protein shows voltage-dependent channel activity with properties similar to those of the purified mitochondrial VDAC or VDAC1/porin 31HL from human B lymphocytes, and its channel activity is completely inhibited by the anion transport inhibitor DIDS and about 80% by DCCD. We also demonstrate the translocation of ATP into the SR lumen and the phosphorylation of the luminal protein sarcalumenin by this ATP. Both ATP translocation and sarcalumenin phosphorylation are inhibited by DIDS, but not by atractyloside, a blocker of the ATP/ADP exchanger. These results indicate the existence of VDAC, thought to be located exclusively in mitochondria, in the SR of skeletal muscle, and its possible involvement in ATP transport. Together with recent studies on VDAC multicompartment location and its dynamic association with enzymes and channels, our findings suggest that VDAC deserves attention and consideration as a protein contributing to various cellular functions.

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

PubMed1996

Ryanodine receptor/calcium release channel conformations as reflected in the different effects of propranolol on its ryanodine binding and channel activity.

1. Propranolol, a beta-blocker, inhibited or stimulated ryanodine binding to both the membrane-bound and purified ryanodine receptor (RyR) depending on the assay conditions. At high NaCl concentrations, propranolol increased the number of ryanodine-binding sites (Bmax) with no effect on the binding affinity. In the presence of 0.2 M NaCl, ryanodine binding was inhibited by propranolol. Half-maximal inhibition was obtained at 1.2 mM and complete inhibition at 2 mM propranolol. The inhibitory effect of propranolol obtained at low NaCl concentration was not restored by increasing the NaCl concentration to 1 M. 2. Modulators of the RyR that are known to alter its conformational states, such as adenine nucleotides, Ca2+ concentration and pH, modified the effect of propranolol on ryanodine binding. In the presence of propranolol and at low NaCl concentrations, ryanodine binding was inhibited and showed no Ca(2+)-, pH-, or time-dependence. 3. Propranolol immediately and completely blocked the channel opening of RyR reconstituted into a planar lipid bilayer. Propranolol-modified non-active channel was reactivated to a subconductive state (about 40% of the control conductance) by ATP. 4. Competition experiments between lidocaine (a stimulatory drug) or tetracaine (an inhibitory drug) and propranolol at 0.2 or 1.0 M NaCl, respectively, suggest the existence of different interaction sites for local anaesthetics and propranolol. 5. These results suggest that propranolol interacts directly with the RyR and modifies its ryanodine binding and single-channel activities. Propranolol effects are altered by the RyR conformational state, suggesting its possible use as a conformational probe for RyR.

Adrenergic beta-Antagonists↗

PubMed1995

Cross-linking of the ryanodine receptor/Ca2+ release channel from skeletal muscle.

The relationship between the tetrameric organization of the ryanodine receptor (RyR) and its activity in binding of ryanodine was approached through cross-linking studies using several bifunctional reagents, differing in their linear dimensions and flexibility, as well as in the reactivity of the active groups. Cross-linking with: 1,5-difluoro-2,4-dinitrobenzene (DFDNB); di(fluoro-3-nitrophenyl)sulfone (DFNPS), 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC); dimethyl suberimidate (DMS); ethylene glycol bis(succinimidylsuccinate) (EGS); and glutaraldehyde resulted in the disappearance of the, 470 kDa, RyR monomer protein band with concomitant appearance of additional bands of molecular masses higher than the monomer. At the relatively low concentrations of the reagents and the conditions used, RyR is the only cross-linked protein of SR membranes. The 'new' protein bands cross-react with antibodies against the RyR and correspond to dimers and tetramers of the RyR subunits while trimers were not detectable. DFDNB and DFNPS produced also a 560 kDa protein band which probably represents an intramolecular cross-linked monomer. The SDS-electrophoretic patterns of the cross-linked purified RyR resemble those of the membrane-bound receptor. Ryanodine binding to the high-affinity site was inhibited by modification of SR membranes with DFDNB and DFNPS, but not with DMS, EDC, EGS and glutaraldehyde, although RyR was completely cross-linked. The inhibition by DFDNB and DFNPS is due to modification of a specific lysyl residue which is also involved in the control of Ca2+ release. On the other hand, cross linking of the RyR with glutaraldehyde or EGS resulted in inhibition of ryanodine binding to the low-affinity, but not to the high-affinity binding sites. Thus, the cross-linking of two or more sites in each monomer (which lead to fixation of dimers or tetramers) did not prevent the conformational changes involved in the binding and occlusion of ryanodine at the high-affinity site, but inhibited its binding to the low-affinity sites.

Animals↗

PubMed1995

Extracellular ATP binding proteins as potential receptors in mucociliary epithelium: characterization using [32P]3'-O-(4-benzoyl)benzoyl ATP, a photoaffinity label.

3'-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [alpha-32P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of 32P incorporation in both proteins on [alpha-32P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca2+ or Mg2+. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP approximately equal to ADP > GTP gamma S > ADP beta S, UTP, 2MeSATP, AMP-PNP > AMP-PCP > AMP > adenosine; for p96 it is: ADP approximately equal to ADP beta S > or = ATP >> AMP-PCP, AMP-PNP > GTP gamma S > or = AMP > 2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADP beta S, UTP > or = 2MeSATP, GTP gamma S, AMP-PNP, ATP > or = ADP > AMP-PCP > adenosine > AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P2 purinoceptor. On the other hand, p96 may represent a P2 purinoceptor or an ectonucleotidase.

Adenosine Triphosphate↗