Activity-dependent translation requires the transport of mRNAs within membraneless protein assemblies known as neuronal granules from the cell body toward synaptic regions. Translation of mRNA is inhibited in these granules during transport but quickly activated in response to neuronal stimuli at the synapse. This raises an important question: how does synaptic activity trigger translation of once-silenced mRNAs? Here, we demonstrate a strong connection between phase separation, the process underlying the formation of many different types of cellular granules, and in vitro inhibition of translation. By using the Fragile X Mental Retardation Protein (FMRP), an abundant neuronal granule component and translational repressor, we show that FMRP phase separates in vitro with RNA into liquid droplets mediated by its C-terminal low-complexity disordered region (i.e., FMRPLCR). FMRPLCRposttranslational modifications by phosphorylation and methylation have opposing effects on in vitro translational regulation, which corroborates well with their critical concentrations for phase separation. Our results, combined with bioinformatics evidence, are supportive of phase separation as a general mechanism controlling activity-dependent translation.

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Propofol (2,6 di-isopropylphenol) is an alkyphenol recently introduced for use as a general anesthetic. The modulation of GABAA receptor activation and desensitization by propofol was studied using a rapid perfusion system and whole-cell voltage-clamp recordings from mouse hippocampal neurons. The effects of concentrations of propofol used clinically on single-channel and synaptic currents were also examined. Propofol evoked current responses (EC50 = 61 microM) and shifted the dose-response curve of GABA-activated current to the left without altering the maximum of the GABA response. Preincubation with propofol and GABA led to desensitization of the GABA response (EC50 = 454 microM and 23 microM, respectively). Saturating concentrations of GABA (600 microM) evoked currents that peaked and then declined in a biexponential fashion with fast and slow time constants of tau f = 1.0 sec and tau s = 3.5 sec. Propofol (10 microM) did not change the amplitude of the peak response but decreased the rates of decay approximately 1.5-fold and enhanced the steady-state current proportionately. Recovery from desensitization was also biexponential (tau f = 11 sec, tau s = 69 sec) but not influenced by propofol. Single-channel recordings from outside-out patches demonstrated that both propofol and GABA activated channels with a 30 pS and 21 pS open state. Propofol increased the frequency but not the duration or conductance of GABA-activated events. Miniature inhibitory postsynaptic currents (mlPSCs) were evoked by the application of hypertonic sucrose to the cell soma. Propofol (2 microM) prolonged the decay time of mlPSCs to an extent similar to which it increased the open probability of GABA-activated channels (2.3- vs 3-fold). A sequential model, based on a previous scheme of GABA receptor gating (Weiss and Magelby, 1989), is presented to summarize propofol's actions on GABAA receptor function. We show through simulation that the model reliably reproduced the whole-cell tracings. Our results indicate that propofol's neurodepressive actions will be associated with enhancement of inhibitory synaptic transmission.

In the mammalian central nervous system, receptors for excitatory amino acid neurotransmitters such as the alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA)-kainate receptor mediate a large fraction of excitatory transmission. Currents induced by activation of the AMPA-kainate receptor were potentiated by agents that specifically stimulate adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase A (PKA) activity or were supported by intracellular application of the catalytic subunit of PKA by itself or in combination with cAMP. Furthermore, depression of these currents by a competitive inhibitor of PKA indicates that AMPA-kainate receptors are regulated by endogenous PKA. Endogenous protein phosphatases also regulate these receptors because an inhibitor of cellular phosphates enhanced kainate currents. Modulation of PKA and phosphatases may regulate the function of these receptors and thus contribute to synaptic plasticity in hippocampal neurons.

1 The effects of propofol (2,6 di-isopropylphenol) on

Phosphorylation of glutamate receptors is probably an important mechanism for modulating excitatory transmission. However, there is little direct evidence to indicate which protein phosphatases can dephosphorylate glutamate or other ligand-gated channels, although it is known that protein phosphatases 1 and 2A play a major part in modulating voltage and second-messenger-gated channels. Here we report that in cultured hippocampal neurons, the N-methyl-D-aspartate (NMDA) receptor can be regulated by endogenous and exogenous serine/threonine protein phosphatases. Phosphatase inhibitors enhanced NMDA currents recorded using the perforated patch technique or in cell-attached patches, whereas protein phosphatases 1 or 2A decreased the open probability of these channels in inside-out patches.

The ability of the constitutively active fragment of protein kinase C (PKM) to modulate N-methyl-D-aspartate (NMDA)-activated currents in cultured mouse hippocampal neurons and acutely isolated CA1 hippocampal neurons from postnatal rats was studied using patch-clamp techniques. The responses of two heterodimeric combinations of recombinant NMDA receptors (NR1a/NR2A and NR1a/NR2B) expressed in human embryonic kidney 293 cells were also examined. Intracellular applications of PKM potentiated NMDA-evoked currents in cultured and isolated CA1 hippocampal neurons. This potentiation was observed in the absence or presence of extracellular Ca2+ and was prevented by the coapplication of the inhibitory peptide protein kinase inhibitor(19-36). Furthermore, the PKM-induced potentiation was not a consequence of a reduction in the sensitivity of the currents to voltage-dependent blockade by extracellular Mg2+. We also found different sensitivities of the responses of recombinant NMDA receptors to the intracellular application of PKM. Some potentiation was observed with the NR1a/NR2A subunits, but none was observed with the NR1a/NR2B combination. Applications of PKM to inside-out patches taken from cultured neurons increased the probability of channel opening without changing single-channel current amplitudes or channel open times. Thus, the activation of protein kinase C is associated with potentiation of NMDA receptor function in hippocampal neurons largely through an increase in the probability of channel opening.

Here we summarize the evidence from two “giant” presynaptic terminals—the squid giant synapse and the mammalian calyx of Held—supporting the involvement of nanodomain calcium signals in triggering of neurotransmitter release. At the squid synapse, there are three main lines of experimental evidence for nanodomain signaling. First, changing the size of the unitary calcium channel current by altering external calcium concentration causes a non-linear change in transmitter release, while changing the number of open channels by broadening the presynaptic action potential causes a linear change in release. Second, low-affinity calcium indicators, calcium chelators, and uncaging of calcium all suggest that presynaptic calcium concentrations are as high as hundreds of micromolar, which is more compatible with a nanodomain type of calcium signal. Finally, neurotransmitter release is much less affected by the slow calcium chelator, ethylene glycol tetraacetic acid (EGTA), in comparison to the rapid chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Similarly, as the calyx of Held synapse matures, EGTA becomes less effective in attenuating transmitter release while the number of calcium channels required to trigger a single fusion event declines. This suggests a developmental transformation of microdomain to nanodomain coupling between calcium channels and transmitter release. Calcium imaging and uncaging experiments, in combination with simulations of calcium diffusion, indicate the peak calcium concentration seen by presynaptic calcium sensors reaches at least tens of micromolar at the calyx of Held. Taken together, data from these provide a compelling argument that nanodomain calcium signaling gates very rapid transmitter release.