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Microdomains of High Calcium Concentration in a Presynaptic Terminal
Abstract
Increases in intracellular calcium concentration are required for the release of neurotransmitter from presynaptic terminals
in all neurons. However, the mechanism by which calcium exerts its effect is not known. A low-sensitivity calcium-dependent
photoprotein (n-aequorin-J) was injected into the presynaptic terminal of the giant squid synapse to selectively detect high
calcium concentration microdomains. During transmitter release, light emission occurred at specific points or quantum emission
domains that remained in the same place during protracted stimulation. Intracellular calcium concentration microdomains on
the order of 200 to 300 micromolar occur against the cytoplasmic surface of the plasmalemma during transmitter secretion,
supporting the view that the synaptic vesicular fusion responsible for transmitter release is triggered by the activation
of a low-affinity calcium-binding site at the active zone.
... 13 C detection offers the possibility to monitor these resonances in detail through simple 2D NMR spectra as illustrated here through the example of the interaction of α-synuclein with calcium ions. Indeed α-synuclein is exposed to Ca 2+ concentration bursts associated with neurotransmitter release [111] and the interaction with calcium ions might also trigger easier aggregation and be linked to the onset of Parkinson's disease. The possibility of monitoring resonances of side chain carboxylate functional groups present in aspartate and glutamate residues is very helpful to reveal the initial steps of the interaction with Ca 2+ [79]. ...
... The lifetime traces in Fig. 3a suggests that CaMKII-NR2B binding is persistent over a 3-h period when calcium is maintained at high levels. Intracellular resting free calcium, the concentration of the fraction of the total calcium in a solution that is ionized and available to activate biological processes, is typically nano-molar, but can transiently reach concentrations in the hundreds of micro-molar in microdomains near the mouths of open calcium channels [48][49][50] . It is not known how stable CaMKII-NR2B interactions are under more physiologically relevant calcium concentrations. ...
Synaptic functions are mediated and modulated by a coordinated choreography of protein conformational changes and interactions in response to intracellular calcium dynamics. Time-lapse Förster resonance energy transfer can be used to study the dynamics of both conformational changes and protein-protein interactions simultaneously under physiological conditions if two resonance energy transfer reactions can be multiplexed. Binary-FRET is a technique developed to independently monitor the dynamics of calcium-calmodulin dependent protein kinase-II catalytic-domain pair separation in the holoenzyme, and its role in establishing activity-dependent holoenzyme affinity for the NR2B binding fragment of the N-methyl-D-aspartate receptor. Here we show that a transient excited-state intermediate exists where paired catalytic-domains in the holoenzyme first separate prior to subsequent NR2B association. Additionally, at non-saturating free calcium concentrations, our multiplexed approach reveals that the holoenzyme exhibits a biochemical form of plasticity, calcium dependent adaptation of T-site ligand binding affinity.
Ca ²⁺ ‐dependent K ⁺ (BK) channels at varicosities in Xenopus nerve–muscle cell cultures were used to quantify experimentally the instantaneous active zone [Ca ²⁺ ] AZ resulting from different rates and durations of Ca ²⁺ entry in the absence of extrinsic buffers and correlate this with neurotransmitter release. Ca ²⁺ tail currents produce mean peak [Ca ²⁺ ] AZ ~ 30 μM; with continued influx, [Ca ²⁺ ] AZ reaches ~45–60 μM at different rates depending on Ca ²⁺ driving force and duration of influx. Both I BK and release are dependent on Ca ²⁺ microdomains composed of both N‐ and L‐type Ca channels. Domains collapse with a time constant of ~0.6 ms. We have constructed an active zone (AZ) model that approximately fits this data, and depends on incorporation of the high‐capacity, low‐affinity fixed buffer represented by phospholipid charges in the plasma membrane. Our observations suggest that in this preparation, (1) some BK channels, but few if any of the Ca ²⁺ sensors that trigger release, are located within Ca ²⁺ nanodomains while a large fraction of both are located far enough from Ca channels to be blockable by EGTA, (2) the I BK is more sensitive than the excitatory postsynaptic current (EPSC) to [Ca ²⁺ ] AZ (K 1/2 –26 μM vs. ~36 μM [Ca ²⁺ ] AZ ); (3) with increasing [Ca ²⁺ ] AZ , the I BK grows with a Hill coefficient of 2.5, the EPSC with a coefficient of 3.9; (4) release is dependent on the highest [Ca ²⁺ ] achieved, independent of the time to reach it; (5) the varicosity synapses differ from mature frog nmjs in significant ways; and (6) BK channels are useful reporters of local [Ca ²⁺ ] AZ .
Calcium (Ca²⁺) signaling is tightly regulated within a presynaptic bouton. Here, we visualize Ca²⁺ signals within hippocampal presynaptic boutons using GCaMP8s tagged to synaptobrevin, a synaptic vesicle protein. We identify evoked presynaptic Ca²⁺ transients (ePreCTs) that derive from synchronized voltage-gated Ca²⁺ channel openings, spontaneous presynaptic Ca²⁺ transients (sPreCTs) that originate from ryanodine sensitive Ca²⁺ stores, and a baseline Ca²⁺ signal that arises from stochastic voltage-gated Ca²⁺ channel openings. We find that baseline Ca²⁺, but not sPreCTs, contributes to spontaneous glutamate release. We employ photobleaching as a use-dependent tool to probe nano-organization of Ca²⁺ signals and observe that all three occur in non-overlapping domains within the synapse at near-resting conditions. However, increased depolarization induces intermixing of these Ca²⁺ domains via both local and non-local synaptic vesicle turnover. Our findings reveal nanosegregation of Ca²⁺ signals within a presynaptic terminal that derive from multiple sources and in turn drive specific modes of neurotransmission.
Heart failure is a serious global health challenge, affecting more than 6.2 million people in the United States and is projected to reach over 8 million by 2030. Independent of etiology, failing hearts share common features, including defective calcium (Ca2+) handling, mitochondrial Ca2+ overload, and oxidative stress. In cardiomyocytes, Ca2+ not only regulates excitation–contraction coupling, but also mitochondrial metabolism and oxidative stress signaling, thereby controlling the function and actual destiny of the cell. Understanding the mechanisms of mitochondrial Ca2+ uptake and the molecular pathways involved in the regulation of increased mitochondrial Ca2+ influx is an ongoing challenge in order to identify novel therapeutic targets to alleviate the burden of heart failure. In this review, we discuss the mechanisms underlying altered mitochondrial Ca2+ handling in heart failure and the potential therapeutic strategies.
Calcium ions (Ca2+) play a critical role in triggering neurotransmitter release. The rate of release is directly related to the concentration of Ca2+ at the presynaptic site, with a supralinear relationship. There are two main sources of Ca2+ that trigger synaptic vesicle fusion: influx through voltage-gated Ca2+ channels in the plasma membrane and release from the endoplasmic reticulum via ryanodine receptors. This chapter will cover the sources of Ca2+ at the presynaptic nerve terminal, the relationship between neurotransmitter release rate and Ca2+ concentration, and the mechanisms that achieve the necessary Ca2+ concentrations for triggering synaptic exocytosis at the presynaptic site.
Synaptotagmin-1 is a vesicular protein and Ca2+ sensor for Ca2+-dependent exocytosis. Ca2+ induces synaptotagmin-1 binding to its own vesicle membrane, called the cis-interaction, thus preventing the trans-interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of the cis- and trans-membrane interaction of synaptotagmin-1 was poorly understood in different Ca2+-buffering conditions. Here we provide an assay to monitor the cis- and trans-membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse the cis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10–100 μM. High PIP2 concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP2 concentrations control the Ca2+-cooperativity of synaptotagmin-1. Our data provide evidence that Ca2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1.
In recent years our understanding of molecular mechanisms of drug action and interindividual variability in drug response has grown enormously. Meanwhile, the practice of anesthesiology has expanded to the preoperative environment and numerous locations outside the OR. Anesthetic Pharmacology: Basic Principles and Clinical Practice, 2nd edition, is an outstanding therapeutic resource in anesthesia and critical care: Section 1 introduces the principles of drug action, Section 2 presents the molecular, cellular and integrated physiology of the target organ/functional system and Section 3 reviews the pharmacology and toxicology of anesthetic drugs. The new Section 4, Therapeutics of Clinical Practice, provides integrated and comparative pharmacology and the practical application of drugs in daily clinical practice. Edited by three highly acclaimed academic anesthetic pharmacologists, with contributions from an international team of experts, and illustrated in full colour, this is a sophisticated, user-friendly resource for all practitioners providing care in the perioperative period.
Citrullination, the post-translational modification of arginine residues, is catalyzed by the four catalytically active peptidylarginine deiminase (PAD or PADI) isozymes and alters charge to affect target protein structure and function. PADs were initially characterized in rodent uteri, and since then, have been described in other female tissues including ovaries, breast, and the lactotrope and gonadotrope cells of the anterior pituitary gland. In these tissues and cells, estrogen robustly stimulates PAD expression resulting in changes in levels over the course of the female reproductive cycle. The best characterized targets for PADs are arginine residues in histone tails, which, when citrullinated, alter chromatin structure and gene expression. Methodological advances have allowed for the identification of tissue-specific citrullinomes, which reveal that PADs citrullinate a wide range of enzymes and structural proteins to alter cell function. In contrast to their important physiological roles, PADs and citrullinated proteins are also involved in several female specific diseases including autoimmune disorders and reproductive cancers. Herein, we review current knowledge regarding PAD expression and function and highlight the role of protein citrullination in both normal female reproductive tissues and associated diseases.
Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+.
Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)
Synapsin I and calcium/calmodulin-dependent protein kinase II were pressure-injected into the preterminal digit of the squid giant synapse to test directly the possible regulation of neurotransmitter release by these substances. Neurotransmitter release was determined by measuring the amplitude, rate of rise, and latency of the postsynaptic potential generated in response to presynaptic depolarizing steps under voltage clamp conditions. Injection of dephosphosynapsin I decreased the amplitude and rate of rise of the postsynaptic potential, whereas injection of either phosphosynapsin I or heat-treated dephosphosynapsin I was without effect. Conversely, injection of calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, increased the rate of rise and amplitude and decreased the latency of the postsynaptic potential. The effects of these proteins were observed without any detectable change in the initial phase of the presynaptic calcium current. A synapsin I-like protein and calcium/calmodulin-dependent protein kinase II were demonstrated by biochemical and immunochemical techniques to be present in squid nervous tissue. The data support the hypothesis that synapsin I regulates the availability of synaptic vesicles for release; we propose that calcium entry into the nerve terminal activates calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, dissociating it from the vesicles and thereby removing a constraint in the release process.
The ultrastructure of the ‘giant synapse’ of the stellate ganglion of the squid was studied with freeze-fracture and thin-sectioning techniques. A sheath of glial cells separates the pre- and post-synaptic axons. At intervals, round-topped processes of the postsynaptic axon pierce the sheath to contact the presynaptic axon. This area of synaptic contact is marked by a widened intercellular cleft containing electron-dense material and by a cluster of synaptic vesicles within the presynaptic cytoplasm. The number of synaptic vesicles in such clusters was greatly reduced by electrical stimulation of the synapse during fixation. Freeze-fracture reveals a roughly circular patch (0.3 μm diameter) of 10 nm particles on the cytoplasmic leaflet of the presynaptic membrane. A similar patch of particles lies on the external leaflet of the apposed postsynaptic membrane.The squid giant synapse thus consists of multiple small pre- and postsynaptic active zones where neurotransmitter is released from the presynaptic terminal and sensed by postsynaptic receptors. Comparison of the structure of these postsynaptic active zones with those at synapses where the transmitter or transmitter action is known suggests that the excitatory transmitter at this synapse is an amino acid.Presumptive gap junctions, marked by particles in the cytoplasmic leaflet, are found between small-diameter axons in the stellate ganglion but not at the giant synapse. Glial-cell membranes contain aggregates of particles and pits suggestive of gap junctions. The aggregates of pits are embedded within linear arrays of particles which somewhat resemble tight junctions.
Quantitative modeling indicates that, in presynaptic terminals, the intracellular calcium concentration profile during inward calcium current is characterized by discrete peaks of calcium immediately adjacent to the calcium channels. This restriction of intracellular calcium concentration suggests a remarkably well specified intracellular architecture such that calcium, as a second messenger, may regulate particular intracellular domains with a great degree of specificity.
We have used a three-dimensional diffusion model of calcium entering the presynaptic nerve terminal through discrete channels to simulate experiments relating transmitter release to presynaptic calcium current. The relationship will be less than linear, or will curve downward, if calcium channels are well separated. It will resemble a power-law function with exponent less than the cooperativity of calcium action if channels are clustered closer together. Large presynaptic depolarizations elicit more release than small depolarizations admitting the same calcium influx. This occurs because large pulses open more channels near each other, with the result that the calcium concentration near release sites is greater, due to overlap of calcium diffusing from adjacent channels.
Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities to Ca2+ concentration. The relative intensity of Ca2+-triggered luminescence of the photoproteins ranged from 0.01 to 190 when compared with natural aequorin (relative intensity 1.0) at pCa 6 for the cases where the relative intensity is less than 1 and at pCa 7 for the cases where the relative intensity is higher than 1. Eight of the semi-synthetic aequorins belonged to the class of e-aequorin. With two of those photoproteins, the degree of dependence of the luminescence intensity ratio I400/I465 on pCa was greater than that with e-aequorin, suggesting that these two photoproteins are possibly superior to e-aequorin in measuring Ca2+ concentration by the ratio method.
Microinjection of aequorin, a bioluminescent protein sensitive tocalcium, into the presynaptic terminal of the squid giant synapse demnonstrated an increase in intracellular calcium ion concentration during repetitive synaptic transmission. Although no light flashes synchronous with individual presynaptic : tion potentials were detected, the results are considered consistent with the hypothesis that entry of calcium into the presynaptic terminal triggers release of e synaptic transmitter substance.