Synaptotagmin-7 Is an Asynchronous Calcium Sensor for Synaptic Transmission in Neurons Expressing SNAP-23

Synchronization of neurotransmitter release with the presynaptic action potential is essential for maintaining fidelity of information transfer in the central nervous system. However, synchronous release is frequently accompanied by an asynchronous release component that builds up during repetitive stimulation, and can even play a dominant role in some synapses. Here, we show that substitution of SNAP-23 for SNAP-25 in mouse autaptic glutamatergic hippocampal neurons results in asynchronous release and a higher frequency of spontaneous release events (mEPSCs). Use of neurons from double-knock-out (SNAP-25, synaptotagmin-7) mice in combination with viral transduction showed that SNAP-23-driven release is triggered by endogenous synaptotagmin-7. In the absence of synaptotagmin-7 release became even more asynchronous, and the spontaneous release rate increased even more, indicating that synaptotagmin-7 acts to synchronize release and suppress spontaneous release. However, compared to synaptotagmin-1, synaptotagmin-7 is a both leaky and asynchronous calcium sensor. In the presence of SNAP-25, consequences of the elimination of synaptotagmin-7 were small or absent, indicating that the protein pairs SNAP-25/synaptotagmin-1 and SNAP-23/synaptotagmin-7 might act as mutually exclusive calcium sensors. Expression of fusion proteins between pHluorin (pH-sensitive GFP) and synaptotagmin-1 or -7 showed that vesicles that fuse using the SNAP-23/synaptotagmin-7 combination contained synaptotagmin-1, while synaptotagmin-7 barely displayed activity-dependent trafficking between vesicle and plasma membrane, implying that it acts as a plasma membrane calcium sensor. Overall, these findings support the idea of alternative syt∶SNARE combinations driving release with different kinetics and fidelity

Regulation of Ca2+ channels by SNAP-25 via recruitment of syntaxin-1 from plasma membrane clusters

SNAP-25 regulates Ca(2+) channels, with potentially important consequences for diseases involving an aberrant SNAP-25 expression level. How this regulation is executed mechanistically remains unknown. We investigated this question in mouse adrenal chromaffin cells and found that SNAP-25 inhibits Ca(2+) currents, with the B-isoform being more potent than the A-isoform, but not when syntaxin-1 is cleaved by botulinum neurotoxin C. In contrast, syntaxin-1 inhibits Ca(2+) currents independently of SNAP-25. Further experiments using immunostaining showed that endogenous or exogenous SNAP-25 expression recruits syntaxin-1 from clusters on the plasma membrane, thereby increasing the immunoavailability of syntaxin-1 and leading indirectly to Ca(2+) current inhibition. Expression of Munc18-1, which recruits syntaxin-1 within the exocytotic pathway, does not modulate Ca(2+) channels, whereas overexpression of the syntaxin-binding protein Doc2B or ubMunc13-2 increases syntaxin-1 immunoavailability and concomitantly down-regulates Ca(2+) currents. Similar findings were obtained upon chemical cholesterol depletion, leading directly to syntaxin-1 cluster dispersal and Ca(2+) current inhibition. We conclude that clustering of syntaxin-1 allows the cell to maintain a high syntaxin-1 expression level without compromising Ca(2+) influx, and recruitment of syntaxin-1 from clusters by SNAP-25 expression makes it available for regulating Ca(2+) channels. This mechanism potentially allows the cell to regulate Ca(2+) influx by expanding or contracting syntaxin-1 clusters

Bumepamine, a brain-permeant benzylamine derivative of bumetanide, does not inhibit NKCC1 but is more potent to enhance phenobarbital's anti seizure efficacy

Correction Volume: 143 Pages: 349-350 DOI: 10.1016/j.neuropharm.2018.10.012Based on the potential role of Na-K-Cl cotransporters (NKCCs) in epileptic seizures, the loop diuretic bumetanide, which blocks the NKCC1 isoforms NKCC1 and NKCC2, has been tested as an adjunct with phenobarbital to suppress seizures. However, because of its physicochemical properties, bumetanide only poorly penetrates through the blood-brain barrier. Thus, concentrations needed to inhibit NKCC1 in hippocampal and neocortical neurons are not reached when using doses (0.1-0.5 mg/kg) in the range of those approved for use as a diuretic in humans. This prompted us to search for a bumetanide derivative that more easily penetrates into the brain. Here we show that bumepamine, a lipophilic benzylamine derivative of bumetanide, exhibits much higher brain penetration than bumetanide and is more potent than the parent drug to potentiate phenobarbital's anticonvulsant effect in two rodent models of chronic difficult-to-treat epilepsy, amygdala kindling in rats and the pilocarpine model in mice. However, bumepamine suppressed NKCC1-dependent giant depolarizing potentials (GDPs) in neonatal rat hippocampal slices much less effectively than bumetanide and did not inhibit GABA-induced Ca2+ transients in the slices, indicating that bumepamine does not inhibit NKCC1. This was substantiated by an oocyte assay, in which bumepamine did not block NKCC1a and NKCC1b after either extra- or intracellular application, whereas bumetanide potently blocked both variants of NKCC1. Experiments with equilibrium dialysis showed high unspecific tissue binding of bumetanide in the brain, which, in addition to its poor brain penetration, further reduces functionally relevant brain concentrations of this drug. These data show that CNS effects of bumetanide previously thought to be mediated by NKCC1 inhibition can also be achieved by a close derivative that does not share this mechanism. Bumepamine has several advantages over bumetanide for CNS targeting, including lower diuretic potency, much higher brain permeability, and higher efficacy to potentiate the anti-seizure effect of phenobarbital.Peer reviewe

TRPing on Cell Swelling-TRPV4 Senses It

The transient receptor potential vanilloid 4 channel (TRPV4) is a non-selective cation channel that is widely expressed and activated by a range of stimuli. Amongst these stimuli, changes in cell volume feature as a prominent regulator of TRPV4 activity with cell swelling leading to channel activation. In experimental settings based on abrupt introduction of large osmotic gradients, TRPV4 activation requires co-expression of an aquaporin (AQP) to facilitate such cell swelling. However, TRPV4 readily responds to cell volume increase irrespectively of the molecular mechanism underlying the cell swelling and can, as such, be considered a sensor of increased cell volume. In this review, we will discuss the proposed events underlying the molecular coupling from cell swelling to channel activation and present the evidence of direct versus indirect swelling-activation of TRPV4. With this summary of the current knowledge of TRPV4 and its ability to sense cell volume changes, we hope to stimulate further experimental efforts in this area of research to clarify TRPV4’s role in physiology and pathophysiology