Role of synaptic proteins in neurotransmitter release-related vesicular trafficking

As is known, regulated exocytosis of synaptic vesicles constitutes a primary means of communication between neurons, and it is subjected to substantial alterations in a number of brain pathologies. Recent investigations showed that vesicular transport events in neuroendocrine cells and presynaptic terminals are realized by a family of specialized membrane proteins of the vesicle (v-SNAREs) and another family located in the target cytoplasmic membrane (t-SNAREs). A variety of such proteins has already been described in different preparations; however, their precise localization and role in vesicular trafficking during functional changes in the cells remain ambiguous. In addition, new synaptic proteins appear to be involved in the vesicular cycle; the functions of these proteins remain unclear. The role of synaptic proteins in the course of cell excitation, in particular functions of core SNARE synaptic proteins (vesicular synaptobrevin/VAMPs and plasma membrane syntaxins/SNAP-25), as well as those of novel presynaptic proteins (Munc-13, Munc-18, CAPS proteins, and others), are discussed in this review. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 155–159, March–April, 2008.     

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains

A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.     

Functional and physical coupling of voltage-sensitive calcium channels with exocytotic proteins: ramifications for the secretion mechanism

The secretion of neurotransmitters is a rapid Ca(2+)-regulated process that brings about vesicle fusion with the plasma membrane. This rapid process (< 100 microseconds) involves multiple proteins located at the plasma and vesicular membranes. Because of their homology to proteins participating in constitutive secretion and protein trafficking, they have been characterized extensively. The sequential events that lead these proteins to vesicle docking and fusion are still unclear. We will review recent studies that demonstrate the operative role played by voltage-sensitive Ca(2+) channels and discuss the relevance for the process of evoked transmitter release. The regulation of Ca(2+) influx by syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and synaptotagmin, and the reciprocity of these proteins in controlling the kinetic properties of the channel will be discussed. Calcium channel and synaptic proteins expressed in Xenopus oocytes demonstrate a strong functional interaction, which could be pertinent to the mechanism of secretion. First, the voltage-sensitive Ca(2+) channels are negatively modulated by syntaxin: this inhibition is reversed by synaptotagmin. Second, the modulation of N-type Ca(2+) channel activation kinetics strongly suggests that the vesicle could be docked at the plasma membrane through direct interaction with synaptotagmin. Finally, these interactions provide evidence for the assembly of the voltage-sensitive Ca(2+) channel with syntaxin 1A, SNAP-25 and synaptotagmin into an excitosome complex: a putative fusion complex with a potential role in the final stages of secretion. Studies suggest that cross-talk between the synaptic proteins and the channel in a tightly organized complex may enable a rapid secretory response to an incoming signal such as membrane depolarization.     

Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca2+ Sensitivity

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca²⁺ activity curve comparable to exocytosis (CV–PM fusion). Here we show that Sr²⁺ and Ba²⁺ also trigger CV–CV fusion, and agents affecting different steps of exocytotic fusion block Ca²⁺, Sr²⁺, and Ba²⁺-triggered CV–CV fusion. The maximal number of active fusion complexes per vesicle, <n\>_Max, was quantified by NEM inhibition of fusion, showing that CV–CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both <n\>_Max and the Ca²⁺ sensitivity of fusion complex activation were comparable to that determined for CV–PM fusion. Using Ca²⁺-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV–CV and CV–PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca²⁺, Sr²⁺, and Ba²⁺, the complex remains Ca²⁺- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca²⁺ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca²⁺-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca²⁺ sensitivity of fusion.     

The time of timing: how Polycomb proteins regulate neurogenesis

The study of mammalian corticogenesis has revealed a critical role for Polycomb group (PcG) factors in timing the execution of developmental choices. Meanwhile, the study of post-translational modifications of PcG factors marks a symmetrical point, namely that the activity of PcG proteins is itself timed in a manner that links progression through the cell cycle to targeting of downstream genes. Finally, in a third symmetrical twist, the studies that dissect the timing of neural fate by Polycomb are also uncovering the importance of timing in the experimental mutation, since ablation of the same PcG member at different developmental stages yields dramatically different results. Here, I weave together these three lines of evidence and develop a unifying model that clarifies the dynamics of Polycomb function in neural development and defines the salient challenges ahead.     

More than just synaptic building blocks: scaffolding proteins of the post-synaptic density regulate dendritic patterning

The dendritic arbor is responsible for receiving and consolidating neuronal input. Outgrowth and morphogenesis of the arbor are complex stages of development that are poorly understood. However, recent findings have identified synaptic scaffolding proteins as novel regulators of these important events. Scaffolding proteins are enriched in the post-synaptic density where they bind and bring into close proximity neurotransmitter receptors, signaling molecules, and regulators of the actin cytoskeleton. This property is important for dendritic spine morphogenesis and maintenance in the mature neuron. Scaffolding proteins are now being described as key regulators of neurite outgrowth, dendritic development, and pattern formation in immature neurons. These proteins, which include post-synaptic-95, Shank and Densin-180, as well as many of their interacting partners, appear to regulate both the microtubule and actin cytoskeleton to influence dendrite morphology. Through a large array of protein-protein interaction domains, scaffolding proteins are able to form large macromolecular complexes that include cytoskeletal motor proteins as well as microtubule and actin regulatory molecules. Together, the new findings form a persuasive argument that scaffolding proteins deliver critical regulatory elements to sites of dendritic outgrowth and branching to modulate the formation and maintenance of the dendritic arbor.     

Current view on regulation of voltage‐gated sodium channels by calcium and auxiliary proteins

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na⁺ channels (Na_V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca²⁺. The molecular and structural details for how Ca²⁺ and/or auxiliary proteins modulate Na_V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca²⁺ acts directly upon the Na_V channel or through interacting proteins, such as the Ca²⁺ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na_V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca²⁺ affects Na_V function, and how altered Ca²⁺‐dependent or FHF‐mediated regulation of Na_V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.     

Redox regulation of calcium ion channels: chemical and physiological aspects

Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease.Changes in the intracellular free Ca²⁺ concentration are key triggers for diverse cellular functions. Ca²⁺ homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca²⁺ permeable ion channels such as voltage-gated (CaV) Ca²⁺ channels, transient receptor potential (TRP) channels and Orai channels.     

Involvement of L-type calcium channels and mitochondria in post-tetanic potentiation: Is it a general rule for different types of synapses?

Our experiments and studies of a few other authors demonstrated that L-type calcium channels and mitochondria are involved in the induction of post-tetanic potentiation (PTP) in a number of preparations (Aplysia central nervous system, hippocampal cell cultures, crayfish neuromuscular junctions, etc.). We extend this conclusion on cortical synapses by the demonstration that inhibitors of mitochondrial Ca²⁺ uptake and release suppress PTP in rat neocortical cell cultures. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 403–404, July–October, 2007.     

Neuronal membrane depolarization and the control of cholinergic muscarinic receptors: selective effect on different neuronal cell types

1. The possibility that a long-lasting neuronal activation regulates the expression of muscarinic cholinergic receptors was studied with three cultured neuronal cell lines. 2. Continuous depolarization of a subclone of the neuroblastoma-glioma NG108-15 hybrid cells with potassium chloride increased by 45-75% the number of cholinergic muscarinic receptors, monitored with 3H-QNB, whereas a short incubation with KCl for 10 min or 6 hr had no effect. 3. The calcium channel blocker verapamil increased the effect of KCl. 4. Two cell lines, named SC9 and WC5, that originate from the rat brain, also bind 3H-QNB. They were therefore used to test whether the effect of chronic depolarization is universal. Depolarized SC9 and WC5 cells, in the presence or absence of verapamil, did not show an increased 3H-QNB binding. 5. Muscarinic receptors of both SC9 and WC5 cells have a higher affinity to pirenzepine than the M-3 receptor subtype of the neuroblastoma-glioma cells, suggesting therefore that the two rat brain cell lines possess M-1 or M-2 receptors. 6. The physiological significance of this differential role of depolarization on the expression of different muscarinic receptors is discussed in the context of their postreceptor second messengers.     

p-CMBS Modifies Extrafacial Sulfhydryl Groups at the Chara Plasma Membrane: Activation of Ca2+ Influx and Inhibition of Two Different K+ Currents

The effect of the membrane impermeant sulfhydryl group (SH) reagent, p-chloromercuribenzenesulfonic acid (p-CMBS), on electrical membrane transport properties of the giant alga, Chara corallina, was determined. In an external medium with a high K⁺ concentration (5 mM) cells typically exhibited stable membrane potentials close to the K⁺equilibrium potential. The steady-state current-voltage (I-V) relation could be dissected into two distinct components: an almost linear ohmic leak current and a voltage-dependent K⁺ current. Adding 0.5 mM p-CMBS to the external medium resulted in an immediate, short depolarization transient (resembling the time course of an action potential) and was associated with a slow down of the cytoplasmic streaming velocity. The depolarization, as well as the streaming inhibition, could be abolished by pretreating cells with the Ca²⁺ channel inhibitor, LaCl₃. This suggests that the depolarization transient reflected a p-CMBS induced Ca²⁺ influx, a scenario known to trigger membrane excitation and slow down of cytoplasmic streaming. From the I-V analysis it appeared that p-CMBS also caused a reversible inhibition of two additional transmembrane currents: (1) a reduction of a leak current and (2) a modification of the deactivation kinetics of the voltage-dependent K⁺ channels. From the I-V difference analysis, the inhibited leak current was identified as a K⁺ current, because the reversal potential was close to the estimated K⁺ equilibrium potential. Control experiments have furthermore shown that the mercapto reagent, dithiothreitol, partly reversed the effect of p-CMBS. This strengthens the view that the action of the mercurial is related to a specific and direct modification of SH groups. The p-CMBS-evoked inhibition of K⁺ currents was not abolished by the LaCl₃ pretreatment, which suggests that the effect of the SH reagent is not induced indirectly by p-CMBS-triggered Ca²⁺ influx. Therefore, it is suggested that the mercurial interacts direcly with the K⁺ transport protein.     

The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.     

Solid-state H and N NMR studies of side-chain and backbone dynamics of phospholamban in lipid bilayers: Investigation of the N27A mutation

Phospholamban (PLB) is an integral membrane protein regulating Ca²⁺ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca²⁺ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with ²H and ¹⁵N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). ²H and ¹⁵N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD₃-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca²⁺-ATPase; thus, showing increased inhibition of Ca²⁺-ATPase.