Purified IP3 receptor from smooth muscle forms an IP3 gated and heparin sensitive Ca2+ channel in planar bilayers.

The IP3 receptor of aortic smooth muscle, purified to near homogeneity, was incorporated into vesicle derived planar bilayers. The receptor forms channels which are gated by Ins(1,4,5)P3 (0.5 microM) and are permeable to Ca2+ (Ca2+ greater than K+ much greater than Cl-). Channel activation is specific for Ins(1,4,5)P3. Essentially no activation of channel currents was found for Ins(1,3,4)P3 or Ins(1,3,4,5)P4 at 10 microM. Heparin (25 micrograms/ml) blocked induced currents completely at all levels of activity while ATP (50 microM) increased mean current levels 2 to 4 fold. Ins(1,4,5)P3 activated mean currents increased non-linearly with voltage above about -40 mV applied voltage. Mean current levels could be reversibly adjusted by voltage to the single channel level (0 to -50 mV) or to macroscopic levels (-50 to -100 mV) over periods exceeding 1 h. Single channel events are characterized by fast transitions between predominantly non-resolved sublevels. Estimates of maximal single event currents yield a slope conductance of 32 +/- 4 pS (0 to -60 mV, 50 mM CaCl2). Thus, the purified IP3 receptor forms a channel with functional properties characteristic of IP3 triggered Ca2+ release.     

Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.     

Biochemical status of renal epithelial Na+ channels determines apparent channel conductance, ion selectivity, and amiloride sensitivity.

Purified bovine renal papillary Na+ channels, when reconstituted into planar lipid bilayers, reside in three conductance states: a 40-pS main state, and two subconductive states (12-13 pS and 24-26 pS). The activity of these channels is regulated by phosphorylation and by G-proteins. Protein kinase A (PKA)-induced phosphorylation increased channel activity by increasing the open state time constants from 160 +/- 30 (main conductance), and 15 +/- 5 ms (both lower conductances), respectively, to 365 +/- 30 ms for all of them. PKA phosphorylation also altered the closed time of the channel from 250 +/- 30 ms to 200 +/- 35 ms, thus shifting the channel into a lower-conductance, long open time mode. PKA phosphorylation increased the PNa:PK of the channel from 7:1 to 20:1, and shifted the amiloride inhibition curve to the right (apparent K(i)amil from 0.7 to 20 microM). Pertussis toxin-induced ADP-ribosylation of either phosphorylated of either phosphorylated or nonphosphorylated channels decreased the PNa:PK to 2:1 and 4:1, respectively, and altered K(i)amil to 8 and 2 microM for phosphorylated and nonphosphorylated channels, respectively. GTP-gamma-S treatment of either phosphorylated or nonphosphorylated channels resulted in an increase of PNa:PK to 30:1 and 10:1, respectively, and produced a leftward shift in the amiloride dose-response curve, altering K(i)amil to 0.5 and 0.1 microM, respectively. These results suggest that amiloride-sensitive renal Na+ channel biophysical characteristics are not static, but depend upon the biochemical state of the channel protein and/or its associated G-protein.     

Gating mechanisms of the type-1 inositol trisphosphate receptor

A large amount of data and observations on inositol 1,4,5-trisphosphate (IP(3)) binding to the IP(3) receptor/Ca(2+) channel, the steady-state activity of the channel, and its inactivation by IP(3) can be explained by assuming one activation and one inhibition module, both allosterically operated by Ca(2+), IP(3), and ATP, and one adaptation element, driven by IP(3), Ca(2+), and the interconversion between two possible conformations of the receptor. The adaptation module becomes completely insensitive to a second IP(3) pulse within 80 s. Observed kinetic responses are well reproduced if, in addition, two module open states are rendered inactive by the current charge carrier Mn(2+). The inactivation time constants are 59 s in the activation, and 0.75 s in the adaptation module. The in vivo open probability of the channel is predicted to be almost in coincidence with the behavior in lipid bilayers for IP(3) levels of 0.2 and 2 microM and one-order-higher at 0.02 microM IP(3), whereas at 180 microM IP(3) the maximal in vivo activity may be 2.5-orders higher than in bilayers and restricted to a narrower Ca(2+) domain (approximately 10 microM-wide versus approximately 100 microM-wide). IP(3) is likely to inhibit channel activity at < or =120 nM Ca(2+) in vivo.     

Stimulation by ATP of Inositol Trisphosphate Accumulation and Calcium Mobilization in Cultured Adrenal Chromaffin Cells

Effects of ATP on accumulation of inositol phosphates and Ca2+ mobilization were investigated in cultured bovine adrenal chromaffin cells. When the cells were stimulated with 30 microM ATP, a rapid and transient rise in intracellular Ca2+ concentration was observed. At the same time, ATP rapidly increased accumulation of inositol phosphates. The concentration-response curve for the ATP-induced Ca2+ mobilization was similar to that for inositol trisphosphate (IP3) accumulation. ATP exerted its maximal effects at 30 microM for either IP3 accumulation or Ca2+ mobilization. The order of the efficacy of the agonists for IP3 accumulation and Ca2+ mobilization at 100 microM was ATP greater than ADP greater than AMP approximately adenosine, AMP (100 microM) and adenosine (300 microM) failed to induce IP3 accumulation and Ca2+ mobilization. Although 100 microM GTP and 100 microM UTP also induced IP3 accumulation and Ca2+ mobilization, their efficacy was less than that of ATP. CTP (100 microM) induced a slight IP3 accumulation, but it did not induce Ca2+ mobilization. Nifedipine (10 microM), a Ca2+ channel antagonist, and theophylline (100 microM), a P1-purinergic receptor antagonist, failed to inhibit the ATP-induced IP3 accumulation and Ca2+ mobilization. The above two cellular responses induced by ATP were also observed in the Ca2+-depleted medium. ATP induced a rapid and transient accumulation of 1,4,5-IP3 (5s), followed by a slower accumulation of 1,3,4-IP3. These results suggest that ATP induces the formation of 1,4,5-IP3 through the P2-purinergic receptor and consequently promotes Ca2+ mobilization from intracellular storage sites in cultured adrenal chromaffin cells.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Molecular determinants of ion permeation and selectivity in inositol 1,4,5-trisphosphate receptor Ca2+ channels

We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca(2+) release channel (inositol 1,4,5-trisphosphate receptors (IP(3)Rs)) that is gated by the second messenger inositol 1,4,5-trisphosphate (IP(3)). To accomplish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a (45)Ca(2+) flux assay and single channel electrophysiological analyses. Two IP(3)R mutations, V2548I and D2550E, retained the ability to release (45)Ca(2+) in response to IP(3). When analyzed at the single channel level; both recombinant channels had IP(3)-dependent open probabilities similar to those observed in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K(+) conductance (489 +/- 13 picosiemens (pS) for V2548I versus 364 +/- 5 pS for wild-type), but retained a Ca(2+) selectivity similar to wild-type channels (P(Ca(2+)):P(K(+)) approximately 4:1). Conversely, D2550E channels were nonselective for Ca(2+) over K(+) (P(Ca(2+)):P(K(+)) approximately 0.6:1), while the K(+) conductance was effectively unchanged (391 +/- 4 pS). These results suggest that amino acid residues Val(2548) and Asp(2550) contribute to the ion conduction pathway. We propose that the pore of IP(3)R channels has two distinct sites that control monovalent cation permeation (Val(2548)) and Ca(2+) selectivity (Asp(2550)).     

GTP gamma S effects on phosphatidylinositol phospholipase C alpha isoenzyme activity isolated from guinea pig uterine smooth muscle at different stages of pregnancy.

The ability of GTP gamma S to activate release of inositol polyphosphates from isolated permeabilised guinea pig uterine smooth muscle cells and from partially purified PI-PLC alpha has been studied. Streptolysin O permeabilised and [3H]inositol prelabelled cells show a time dependent release of inositol polyphosphates, predominantly inositol 4-phosphate. Ca2+ stimulated IP release with a Ka of 161 +/- 1.1 nM and this was further enhanced in an additive manner by GTP gamma S between 1-100 microM; the Ka for Ca2+ in the presence of 0.1 mM GTP gamma S was 117 +/- 0.7 nM. GTP gamma S activation of IP production did not require Ca2+ in the medium. Permeabilisation of the uterine smooth muscle cells with Streptolysin O readily released PI-PLC activity into the medium. However, unlike studies with isolated membranes 63.4 +/- 6.4% of the enzyme activity remained associated with membranes and/or particulate fractions of the cell. Studies were undertaken with PI-PLC alpha, the predominant isoenzyme form, partially purified from uterine smooth muscle at different stages of pregnancy by Q-Sepharose and Heparin-Agarose chromatography. The enzyme co-purifies with firmly associated GTP-binding activity. Enzyme prepared from near-term uterus is activated by 0.1 mM GTP gamma S, up to 100% when Ca2+ is between 0.1-1 microM, while 10 microM AlF4- under those conditions caused complete inhibition of the enzymes. Responses for enzymes prepared from non-pregnant uteri were broadly similar. In contrast enzyme preparations from guinea pig uteri at 20-60 days of pregnancy show an inhibition of activity in response to 0.1 mM GTP gamma S addition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium uptake-dependent and -independent mechanisms of inositol trisphosphate formation in adrenal chromaffin cells: comparative studies with high K+, carbamylcholine and angiotensin II

When [3H]inositol prelabelled cultured bovine adrenal chromaffin cells were stimulated with 56 mM KCl (high K+), 300 microM carbamylcholine (CCh) or 10 microM angiotensin II (Ang II), a rapid accumulation of [3H]IP3 was observed. At the same time, high K+ or CCh induced rapid increases in 45Ca2+ uptake, but Ang II did not induce a significant 45Ca2+ uptake. The concentration-response curve for KCl-induced [3H]IP3 accumulation coincided well with that for KCl-induced 45Ca2+ uptake into the cells. Nifedipine, a Ca2+ channel antagonist, inhibited the high K(+)-induced [3H]IP3 accumulation and 45Ca2+ uptake with a similar potency. Nifedipine at a similar concentration range also inhibited CCh-induced 45Ca2+ uptake. Although nifedipine inhibited CCh-induced [3H]IP3 accumulation, the potency was approximately 300-fold less than that for the inhibition of 45Ca2+ uptake. Nifedipine failed to affect the Ang II-induced [3H]IP3 accumulation. BAY K 8644 (2 microM), a Ca2+ channel activator, plus partially depolarizing concentration of KCl (14 mM), induced 45Ca2+ uptake and [3H]IP3 accumulation. Ionomycin (1 microM and 10 microM), a Ca2+ ionophore, also induced 45Ca2+ uptake and [3H]IP3 accumulation in a concentration-dependent manner. Pretreatment of the cells with protein kinase C activator, 100 nM 12-O-tetradecanoyl phorbol-13-acetate, for 10 min, partially inhibited CCh and Ang II-induced [3H]IP3 accumulation, but failed to inhibit the high K(+)-induced accumulation. Furthermore, the effects of high K+ and Ang II on the IP3 accumulation was additive. Ang II and CCh induced a rapid and transient increase in inositol 1,4,5-trisphosphate (1,4,5-IP3) accumulation (5 s) followed by a slower accumulation of inositol 1,3,4-trisphosphate (1,3,4-IP3). High K+ evoked an increase in 1,3,4-IP3 accumulation but obvious accumulation of 1,4,5-IP3 could not be detected. In Ca2(+)-depleted medium, high K(+)-induced [3H]IP3 accumulation was completely abolished, whereas [3H]IP3 accumulation induced by CCh and Ang II was partially inhibited. These results demonstrate the existence of the Ca2+ uptake-triggered mechanism of IP3 accumulation represented by high K+, and also the Ca2+ uptake-independent mechanism of IP3 accumulation represented by Ang II in cultured bovine adrenal chromaffin cells. Mechanism of CCh-induced IP3 accumulation has an intermediate property between those of high K+ and Ang II.     

Ca2+ release by inositol-trisphosphorothioate in isolated triads of rabbit skeletal muscle.

The effectiveness of the nonmetabolizable second messenger analogue DL-myo-inositol 1,4,5-trisphosphorothioate (IPS3) described by Cooke, A. M., R. Gigg, and B. V. L. Potter, (1987b. Jour. Chem. Soc. Chem. Commun. 1525-1526.) was examined in triads purified from rabbit skeletal muscle. A Ca2+ electrode uptake-release assay was used to determine the size and sensitivity of the IPS3-releasable pool of Ca2+ in isolated triads. Uptake was initiated by 1 mM MgATP, pCa 5.8, pH 7.5 Release was initiated when the free Ca2+ had lowered to pCa approximately 7. We found that 5-25 microM myo-inositol 1,4,5-trisphosphate (IP3), and separately IPS3, consistently released 5-20% of the Ca2+ pool actively loaded into triads. Single channel recording was used to determine if ryanodine receptor Ca2+ release channels were affected by IPS3 at the same myoplasmic Ca2+ and IPS3 concentrations. Open probability of ryanodine receptor Ca2+ release channels was monitored in triads fused to bilayers over long periods (200 s) in the absence and following addition of 30 microM IPS3 to the same channel. At myoplasmic pCa approximately 7, IPS3 had no effect in the absence of MgATP (Po = 0.0094 +/- 0.001 in control and Po = 0.01 +/- 0.006 after IPS3) and slightly increased activity in the presence of 1 mM MgATP (Po = 0.024 +/- 0.03 in control and Po = 0.05 +/- 0.03 after IPS3). Equally small effects were observed at higher myoplasmic Ca2+. The onset of channel activation by IPS3 or IP3 was slow, on the time scale 20-60 s. We suggest that in isolated triads of rabbit skeletal muscle, IP3-induced release of stored Ca2+ is probably not mediated by the opening of Ca2+ release channels.     

Activation of phospholipase C by the agonist U46619 is inhibited by cromakalim-induced hyperpolarization in porcine coronary artery.

We measured inositol 1,4,5-trisphosphate (IP3) production, intracellular calcium concentration ([Ca2+]i) and force of contraction induced by a thromboxane A2 analogue U46619 in porcine coronary artery to elucidate the relaxant effect of a K+ channel opener cromakalim. Cromakalim (10 microM) significantly inhibited the production of IP3, Ca2+ release from intracellular stores and contraction induced by 300 nM U46619. The inhibitory effect of cromakalim on IP3 was blocked by a K+ channel blocker tetrabutylammonium (TBA, 3 mM) and counteracted by 20 mM KCl-induced depolarization. These results suggest that the hyperpolarization of the plasma membrane by cromakalim inhibits the activation of phospholipase via the stimulation of the thromboxane A2 receptor to result in vasodilation.     

K+-selective channel from sarcoplasmic reticulum of split lobster muscle fibers

The patch clamp technique has been used to study channels in a membrane inside a cell. A single muscle fiber is skinned in relaxing saline (high K+, low Ca2+ with EGTA and ATP), leaving the native sarcoplasmic reticulum (SR) membrane exposed for patching. Fibers are dissected from the second antenna remotor muscles of the American lobster, Homarus americanus. Transmission and scanning electron microscopy confirm the large volume fraction of SR (approximately 70%) and absence of sarcolemma in this unusual skinned preparation. The resting potential of the SR was measured after the resistance of the patch of membrane was broken down. It is near 0 mV (-0.4 +/- 0.6 mV). The average input resistance of the SR is 842 +/- 295 M omega. Some 25% of patches contain a K+-selective channel with a mean open time of seconds and the channel displays at least two conducting states. The open probability is weakly voltage dependent, large at zero and positive potentials (cytoplasm minus SR lumen), and decreasing at negative potentials. The maximal conductance of this channel is 200 +/- 1 pS and the substate conductance is 170 +/- 3 pS in symmetrical 480 mM K+ solution. The current-voltage relation of the open channel is linear over a range of +/- 100 mV. The selectivity is similar to the SR K+ channel of vertebrates: PK/PNa is 3.77 +/- 0.03, determined from reversal potential measurements, whereas gamma K/gamma Na is 3.28 +/- 0.06, determined from open-channel conductance measurements in symmetrical 480 mM solutions. Voltage-dependent block in the lobster SR K+ channel is similar to, but distinct from, that reported for the vertebrate channels. It occurs asymmetrically when hexamethonium is added to both sides of the membrane. The block is more effective from the cytoplasmic side of the channel.     

The bell-shaped Ca2+ dependence of the inositol 1,4, 5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin.

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 microM if the cytosolic Ca2+ concentration was 0.3 microM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 microM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 microM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.     

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.     

Muscarinic Cholinergic Receptor-Mediated Phosphoinositide Metabolism in Peripheral Nerve

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.     

Bradykinin induces a rapid release of inositol trisphosphate from a neuroblastoma hybrid cell line NCB-20 that is not antagonized by enkephalin

In mouse neuroblastoma x Chinese hamster brain clonal cell line NCB-20, bradykinin (BK) receptor stimulation causes phosphoinositide hydrolysis and release of inositol phosphates. Maximum stimulation (4-fold) of [2-3H]inositol trisphosphate (IP3) release in the absence of Li+ from NCB-20's prelabelled for 20-24 hours with [2-3H]myo-inositol (15 microCi/confluent 60mm dish) occurred after 5-10 seconds of bradykinin exposure, with an EC50 of approximately 100nM. Inositol bisphosphate (IP2) and inositol monophosphate (IP1) also showed increases (2.9-fold and 1.5 fold, respectively), with peaks at 15-20 seconds and 50 seconds, respectively. Under these same conditions, D-Ala2-D-Leu5 enkephalin (DADLE) (10 microM), an opiate agonist with 2nM affinity, gave no stimulation of IP3 release. Furthermore, it did not block BK-initiated release, both when applied simultaneously with BK and when cells were preincubated with DADLE for 100 minutes to lower cyclic AMP levels. These results show that pain-inducing BK has a major acute stimulatory effect on receptor-phospholipase C-coupled IP3 release, the opioid peptide DADLE has no such effect and, DADLE does not block the IP3 release induced by BK.     

Purified ryanodine receptor from rabbit skeletal muscle is the calcium- release channel of sarcoplasmic reticulum

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.     

Three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor at 24 A resolution

We report here the first three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R). From cryo-electron microscopic images of purified receptors embedded in vitreous ice, a three-dimensional structure was determined by use of standard single particle reconstruction techniques. The structure is strikingly different from that of the ryanodine receptor at similar resolution despite molecular similarities between these two calcium release channels. The 24 A resolution structure of the IP(3)R takes the shape of an uneven dumbbell, and is approximately 170 A tall. Its larger end is bulky, with four arms protruding laterally by approximately 50 A and, in comparison with the receptor topology, probably corresponds to the cytoplasmic domain of the receptor. The lateral dimension at the height of the protruding arms is approximately 155 A. The smaller end, whose lateral dimension is approximately 100 A, has structural features indicative of the membrane-spanning domain. A central opening in this domain, which is occluded on the cytoplasmic half, outlines a pathway for calcium flow in the open state of the channel.     

Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.