The inositol 1,4,5-trisphosphate (InsP3) receptor.
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Adenosine Triphosphate↗
Biomedical subjects
B E Ehrlich
Publications and source records attributed to B E Ehrlich.
Functional properties of intracellular calcium-release channels.
Two major classes of intracellular calcium-release channels have been identified, the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. These channels are the largest ion channels identified to date. Recent studies have established that approximately 90% of each of these proteins protrudes into the cytoplasm, presumably exposing many regulatory sites on the channel and allowing functional interactions with other cytoplasmic proteins. Current work is aimed at understanding the molecular mechanisms and cellular roles of these regulatory processes.
Animals↗
Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein.
FK506-binding protein (FKBP12) was originally identified as the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. The cellular function of FKBP12, a ubiquitously expressed 12,000-dalton proline isomerase, has been unknown. FKBP12 copurifies with the 565,000-dalton ryanodine receptor (RyR), four of which form intracellular Ca2+ release channels of the sarcoplasmic and endoplasmic reticula. By coexpressing the RyR and FKBP12 in insect cells, we have demonstrated that FKBP12 modulates channel gating by increasing channels with full conductance levels (by > 400%), decreasing open probability after caffeine activation (from 0.63 +/- 0.09 to 0.04 +/- 0.02), and increasing mean open time (from 4.4 +/- 0.6 ms to 75 +/- 41 ms). FK506 or rapamycin, inhibitors of FKBP12 isomerase activity, reverse these stabilizing effects. These results provide the first natural cellular function for FKBP12, and establish that the functional Ca2+ release channel complex includes FKBP12.
Animals↗
The pharmacology of intracellular Ca(2+)-release channels.
Two classes of intracellular Ca(2+)-release channels, the ryanodine receptor and the inositol (1,4,5)-trisphosphate (IP3) receptor, are essential for spatio-temporal Ca2+ signalling in cells. Heparin and caffeine have been widely used to study these channels. It was originally thought that caffeine acts solely as an agonist for the ryanodine receptor and heparin acts solely as an inhibitor for the IP3 receptor. However, recent experiments indicate that these compounds have multiple effects, and are discussed in this review by Barbara Ehrlich and colleagues. In the same concentration range, caffeine activates the ryanodine receptor and inhibits the IP3 receptor, and heparin inhibits the IP3 receptor and activates the ryanodine receptor. More specific pharmacological tools that are suitable for studies of ryanodine and IP3 receptors are now beginning to emerge.
Animals↗
Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium.
The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single-channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single-ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.
Animals↗
Caffeine-induced inhibition of inositol(1,4,5)-trisphosphate-gated calcium channels from cerebellum.
Effects of the xanthine drug caffeine on inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels from canine cerebellum were studied using single channels incorporated into planar lipid bilayers. Caffeine, used widely as an agonist of ryanodine receptors, inhibited the activity of InsP3-gated Ca channels in a noncooperative fashion with half-inhibition at 1.64 mM caffeine. The frequency of channel openings was decreased more than threefold after addition of 5 mM caffeine; there was only a small effect on mean open time of the channels, and the single channel conductance was unchanged. Increased InsP3 concentration overcame the inhibitory action of caffeine, but caffeine did not reduce specific [3H]InsP3 binding to the receptor. The inhibitory action of caffeine on InsP3 receptors suggests that the action of caffeine on the intracellular Ca pool must be interpreted with caution when both ryanodine receptors and InsP3 receptors are present in the cell.
Animals↗
Intracellular calcium release channels.
Two classes of intracellular calcium release channels have been identified, the ryanodine receptor and the inositol 1,4,5-trisphosphate (InsP3) receptor. Characterizations of each receptor at the single channel level has shown that the channel types are functionally distinct. These two channels differ in the relative density and localization within the cell, in the mechanism of activation, in the regulation by Ca and ATP, and the sensitivity to pharmacological agents. The differences in the properties and regulation of the two types of intracellular calcium release channels are important for determining cell-specific responses to stimuli and allows the cell to adjust Ca signalling to the needs of the physiological state of the cell.
Adenosine Triphosphate↗
ATP modulates the function of inositol 1,4,5-trisphosphate-gated channels at two sites.
The inositol 1,4,5-trisphosphate (IP3) receptor, a Ca(2+)-permeable channel, plays a key role in intracellular Ca2+ signaling. The effects of ATP on the IP3 receptor at the single-channel level were characterized after channel incorporation into planar lipid bilayers. ATP alone was not sufficient to open the IP3-gated channel, but addition of ATP or nonhydrolyzable ATP analogs in the presence of IP3 increased the frequency of channel openings 4.8-fold and increased the average duration of channel openings 2.5-fold; channel conductance was unchanged. High concentrations of ATP (> 4 mM) decreased channel activity most probably by competing with IP3-binding site. Allosteric modulation of IP3-induced Ca2+ release by ATP may contribute to the maintenance of cell viability during periods of energy starvation.
Adenosine Triphosphate↗
Activation of the calcium release channel (ryanodine receptor) by heparin and other polyanions is calcium dependent.
Heparin has been used as a potent competitive inhibitor of inositol 1,4,5-trisphosphate (IP3)-binding to IP3 receptors and to block IP3-gated calcium channels in bilayer experiments. In contrast to the effect on the IP3-gated channel, heparin (0.1-1 micrograms/ml) opened the Ca release channel (ryanodine receptor). Other polyanions such as pentosan polysulfate and polyvinyl sulfate also activated the Ca release channel. The effect of polyanions on the Ca release channel was Ca dependent. Polyanion addition activated the Ca release channel when free Ca was > 80 nM, but was ineffective when free Ca was < 20 nM. The level of channel activation could be altered by manipulating the free Ca concentration. These results suggest that the polyanions act by increasing the local concentration of Ca near regulatory sites on the channel complex. As most cells have both types of intracellular channels, the opposite effects of the polyanions on the two channel types suggests that addition of polyanions to intact cells may produce multiple effects.
Animals↗
Guanine nucleotides modulate calcium currents in a marine Paramecium.
Voltage-dependent calcium channels play a critical role in many cell functions and in many cell types ranging from protozoa to vertebrates. We have shown previously that guanine nucleotides modulate the calcium action potential and the duration of backward swimming in Paramecium, both indirect measurements of calcium channel function. To determine whether guanine nucleotides to indeed alter calcium currents, the inward calcium current (ICa) in Paramecium calkinsi was studied. First, the calcium current was characterized. The magnitude of ICa increased as the extracellular calcium concentration was increased from 0.5 to 50 mmol l-1, unlike the situation in freshwater species of Paramecium where the inward calcium current magnitude is maximal when extracellular calcium levels reach 1 mmol l-1. Inorganic compounds (NiCl2 at 10 mumol l-1 and CdCl2 at 1 mmol l-1) and organic compounds (naphthalene sulfonamides, W-7 and W-12-Br at 100 and 2 mumol l-1, respectively) reduced ICa. Regardless of the holding membrane potential (from -80 to -20 mV), the threshold activation for ICa was at -10 mV and the maximum value of ICa was reached at +20 mV, suggesting that there is only one type of calcium channel in P. calkinsi. Second, we injected GTP gamma S, GTP and GDP beta S into voltage-clamped cells while monitoring calcium and/or potassium currents. GTP gamma S increased the magnitude of ICa by 42 +/- 6% (mean +/- S.D., N = 5) and the effect was irreversible, GTP increased the magnitude of ICa by 37 +/- 4% (N = 4) in a reversible manner, and GDP beta S decreased ICa by 57 +/- 8% (N = 3) irreversibly. The outward potassium currents did not change when GTP gamma S was injected into the cells. These results support the hypothesis that injection of guanine nucleotides modulates the voltage-dependent calcium channel in P. calkinsi, presumably by activating G-protein-dependent processes.
Animals↗
Planar lipid bilayers on patch pipettes: bilayer formation and ion channel incorporation.
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Electrophysiology↗
Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum.
Release of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel and a calcium-gated channel (ryanodine receptor). Using specific antibodies, both receptors were found in Purkinje cells of cerebellum. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 microM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 microM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feedback and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.
Adenosine Triphosphate↗
Regional differences in calcium-release channels from heart.
The heart is a heterogeneous tissue composed of several cell types tailored for specialized functions. We found that intracellular channels also exhibit regional specialization. In cardiac and skeletal muscle these channels are called the calcium-release channel and are identified by activation with either calcium or caffeine and inhibition by the hexavalent cation ruthenium red. The calcium-release channel of the sarcoplasmic reticulum from the interventricular septum has a smaller conductance (31 pS vs. 100 pS) and has longer open and closed times when compared with the channel from left-ventricular free wall. An additional calcium-permeable channel with an even smaller conductance (17 pS) was found in the septum, and this channel is similar to the inositol 1,4,5-trisphosphate-gated channel from smooth muscle and different from the calcium-release channel (ryanodine receptor) from skeletal and cardiac muscle. The inositol 1,4,5-trisphosphate-activated channel may be derived from specialized conducting tissue that is relatively abundant in the septum, whereas the other calcium-release channels may be derived from regionally specialized myocardial cells in the septum and free wall.
Animals↗
Comparison of calcium release from sarcoplasmic reticulum of slow and fast twitch muscles.
The mechanism of Ca2+ release from the sarcoplasmic reticulum (SR) of slow and fast twitch muscle was compared by examining biochemical characteristics, ryanodine binding, Ca2+ efflux, and single Ca2+ channel properties of SR vesicles. Although many features of the Ca2+ release channel were comparable, two functional assays revealed remarkable differences. The comparable properties include: a high molecular weight protein from both types of muscle was immunologically equivalent, and Scatchard analysis of [3H]ryanodine binding to SR showed that the Kd was similar for slow and fast SR. In the flux assay the sensitivity to the agonists caffeine, doxorubicin, and Ca2+ and the antagonists Mg2+, ruthenium red, and tetracaine differed only slightly. When SR vesicles were incorporated into lipid bilayers, the single-channel conductances of the Ca2+ release channels were indistinguishable. The distinguishing properties are: When Ca2+ release from passively 45Ca(2+)-loaded SR were monitored by rapid filtration, the initial rates of Ca2+ release induced by Ca2+ and caffeine were three times lower in slow SR than in fast SR. Similarly, when Ca2+ release channels were incorporated into lipid bilayers, the open probability of the slow SR channel was markedly less, mainly due to a longer mean closed time. Our results indicate that slow and fast muscle have ryanodine receptors that are biochemically analogous, yet functional differences in the Ca2+ release channel may contribute to the different time to peak contraction observed in intact slow and fast muscles.
Animals↗
GTP-gamma-S increases the duration of backward swimming behavior and the calcium action potential in marine Paramecium.
Behavioral and electrophysiological experiments were made to examine the hypothesis that G-proteins modulate the voltage-dependent calcium channel in the marine ciliate Paramecium calkinsi. It was found that guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), an analogue of GTP that binds to and activates G-proteins, increased the duration of backward swimming behavior in reversibly permeabilized Paramecium in an irreversible and concentration-dependent manner. At 1 mumol l-1 GTP-gamma-S, the duration of backward swimming behavior was increased fivefold. Other nucleotides and related compounds did not have a significant effect on the backward swimming behavior. To evaluate whether the behavioral effects were due to ion channel modulation, the calcium action potential in intact Paramecium was monitored before and after guanine nucleotide injection. Within 5 min after the injection of GTP-gamma-S or GTP into the cell, the duration of the calcium action potential was prolonged at least threefold. Like the behavioral response, the GTP-gamma-S effect on the calcium action potential duration was irreversible, whereas the effect of GTP began to decay after 6 min. GDP-beta-S, which binds to and inactivates G-proteins, markedly reduced the calcium action potential within 5 min after injection. These results support the hypothesis that the voltage-dependent calcium channels present in Paramecium are modulated by GTP-binding proteins.
Action Potentials↗
Inositol 1,4,5-trisphosphate-gated channels in cerebellum: presence of multiple conductance states.
The mechanism by which inositol 1,4,5-triphosphate (InsP3) induces calcium (Ca) release from the reticulum of canine cerebellum was examined. Reticular membrane vesicles used in these experiments accumulated Ca in the presence of ATP and then released approximately 30% of the accumulated Ca upon addition of micromolar concentrations of InsP3. When these membrane vesicles were incorporated into planar lipid bilayers, InsP3-gated Ca channels were observed. Up to four current amplitudes were observed at a given voltage, yielding conductances of 20, 40, 60, and 80 pS with 50 mM Ca as the current carrier. Thus, the cerebellar InsP3-gated Ca channel exhibits four conductance levels that are multiples of a unit conductance step. Moreover, examination of the single-channel records showed both openings directly to each of the current levels and rapid transitions between current levels. These four conductance steps may reflect the interaction among the four InsP3 receptors thought to comprise the InsP3-gated Ca channel in these tissues. Examination of the InsP3 dependence of channel openings and Ca release from vesicles, however, yielded Hill coefficients of 1-1.3. Thus, we hypothesize that it takes only one molecule of InsP3 to open the channel. The observation that the conductance of the InsP3-gated Ca channel assumes four levels that are multiples of a unit conductance suggests that the number of interacting InsP3 receptors in one complex can vary from one to four and supports the hypothesis that the channel is a tetramer.
Animals↗
Biphasic effects of doxorubicin on the calcium release channel from sarcoplasmic reticulum of cardiac muscle.
To define the mechanism of doxorubicin cardiotoxicity, the effects of doxorubicin and caffeine were examined on calcium release channels from cardiac sarcoplasmic reticulum. We found that calcium release from cardiac sarcoplasmic reticulum vesicles was induced by both compounds. When sarcoplasmic reticulum vesicles were incorporated into planar lipid bilayers, calcium-permeable channels were observed. Addition of caffeine (2.5-10 mM) increased channel open probability from less than 0.1% to 40%, and this effect persisted for a mean of 44 minutes. In contrast, doxorubicin (2.5-10 microM) had a biphasic effect; initially, doxorubicin activated the channel, whereas after a mean of 8 minutes, the channel became irreversibly inhibited. Although the degree of channel activation by doxorubicin was concentration dependent, the time needed to inactivate the channel was concentration independent. Pretreatment with dithiothreitol (0.2 mM) prevented doxorubicin-induced channel inactivation, and channel activity persisted for an average of 58 minutes. Dithiothreitol alone did not alter channel open probability. Our results support the hypotheses that 1) the integrity of sulfhydryl groups is important for some aspects of calcium release channel function and 2) activation and inactivation of the channel are separable processes. The biphasic effect of doxorubicin on channel function may also correspond to the clinically observed adverse effects of doxorubicin, a widely used chemotherapeutic agent that, after prolonged usage, causes a dilated cardiomyopathy.
Animals↗
Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum.
Inositol 1,4,5-trisphosphate (InsP3) can initiate calcium release into the cytoplasm in a variety of cells. From experiments using permeabilized cells, membrane vesicles, and patch-clamp techniques, it has been suggested that InsP3 acts by directly opening calcium channels. Here, we show that InsP3 induced openings of channels in planar lipid bilayers into which vesicles made from aortic muscle sarcoplasmic reticulum (SR) were incorporated. Activation of channels by InsP3 was not observed when vesicles made from SR of cardiac or skeletal muscle were incorporated into planar lipid bilayers. The present study demonstrates for the first time unique properties of an InsP3-gated calcium channel in sarcoplasmic reticulum vesicles from vascular smooth muscle. This InsP3-activated channel from aortic SR differs strikingly from the calcium-gated calcium channel of striated muscle SR in single-channel conductance and pharmacology.
Animals↗