Beyond direct synaptic communication, neurons are able to talk to each other without making synapses. They are able to send chemical messages by means of diffusion to target cells via the extracellular space, provided that the target neurons are equipped with high-affinity receptors. While synaptic transmission is responsible for the 'what' of brain function, the 'how' of brain function (mood, attention, level of arousal, general excitability, etc.) is mainly controlled non-synaptically using the extracellular space as communication channel. It is principally the 'how' that can be modulated by medicine. In this paper, we discuss different forms of non-synaptic transmission, localized spillover of synaptic transmitters, local presynaptic modulation and tonic influence of ambient transmitter levels on the activity of vast neuronal populations. We consider different aspects of non-synaptic transmission, such as synaptic-extrasynaptic receptor trafficking, neuron-glia communication and retrograde signalling. We review structural and functional aspects of non-synaptic transmission, including (i) anatomical arrangement of non-synaptic release sites, receptors and transporters, (ii) intravesicular, intra-and extracellular concentrations of neurotransmitters, as well as the spatiotemporal pattern of transmitter diffusion. We propose that an effective general strategy for efficient pharmacological intervention could include the identification of specific non-synaptic targets and the subsequent development of selective pharmacological tools to influence them.
BackgroundThere is only one established drug binding site on sodium channels. However, drug binding of sodium channels shows extreme promiscuity: ∼25% of investigated drugs have been found to potently inhibit sodium channels. The structural diversity of these molecules suggests that they may not share the binding site, and/or the mode of action. Our goal was to attempt classification of sodium channel inhibitors by measuring multiple properties of inhibition in electrophysiology experiments. We also aimed to investigate if different properties of inhibition correlate with specific chemical properties of the compounds.Methodology/Principal FindingsA comparative electrophysiological study of 35 compounds, including classic sodium channel inhibitors (anticonvulsants, antiarrhythmics and local anesthetics), as well as antidepressants, antipsychotics and neuroprotective agents, was carried out using rNav1.2 expressing HEK-293 cells and the QPatch automatic patch-clamp instrument. In the multi-dimensional space defined by the eight properties of inhibition (resting and inactivated affinity, potency, reversibility, time constants of onset and offset, use-dependence and state-dependence), at least three distinct types of inhibition could be identified; these probably reflect distinct modes of action. The compounds were clustered similarly in the multi-dimensional space defined by relevant chemical properties, including measures of lipophilicity, aromaticity, molecular size, polarity and electric charge. Drugs of the same therapeutic indication typically belonged to the same type. We identified chemical properties, which were important in determining specific properties of inhibition. State-dependence correlated with lipophilicity, the ratio of the neutral form of molecules, and aromaticity: We noticed that the highly state dependent inhibitors had at least two aromatic rings, logP>4.0, and pKa<8.0.Conclusions/SignificanceThe correlations of inhibition properties both with chemical properties and therapeutic profiles would not have been evident through the sole determination of IC50; therefore, recording multiple properties of inhibition may allow improved prediction of therapeutic usefulness.
Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the “steady-state slow inactivation curve”), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated.
The effect of monoamine uptake inhibitor-type antidepressants on sodium channels of hippocampal neurons was investigated. Members of the tricyclic group of antidepressants are known to modify multiple targets, including sodium channels, whereas selective serotonin-reuptake inhibitors (SSRIs) are regarded as highly selective compounds, and their effect on sodium channels was not investigated in detail. In this study, a representative member of each group was chosen: the tricyclic antidepressant desipramine and the SSRI fluoxetine. The drugs were roughly equipotent use-dependent inhibitors of sodium channels, with IC 50 values ϳ100 M at Ϫ150 mV holding potential, and ϳ1 M at Ϫ60 mV. We suggest that therapeutic concentrations of antidepressants affect neuronal information processing partly by direct, activity-dependent inhibition of sodium channels. As for the mechanism of inhibition, use-dependent inhibition by antidepressants was believed to be due to a preferential affinity to the fast-inactivated state. Using a voltage and perfusion protocol by which relative affinities to fast-versus slow-inactivated states could be assessed, we challenged this view and found that the affinity of both drugs to slowinactivated state(s) was higher. We propose a different mechanism of action for these antidepressants, in which slow rather than fast inactivation plays the dominant role. This mechanism is similar but not equivalent with the novel mechanism of usedependent sodium channel inhibition previously described by our group (Neuroscience
The rat α7 nicotinic acetylcholine receptor (nAChR) has a proline residue near the middle of the β9 strand. The replacement of this proline residue at position 180 (P180) by either threonine (α7-P180T) or serine (α7-P180S) slowed the onset of desensitization dramatically, with half-times of ∼930 and 700 ms, respectively, compared to 90 ms for the wild-type receptor. To investigate the importance of the hydroxyl group on the position 180 side-chains, the mutant receptors α7-P180Y and α7-P180F were studied and showed half-times of desensitization of 650 and 160 ms, respectively. While a position 180 side-chain OH group may contribute to the slow desensitization rates, α7-P180S and α7-P180V resulted in receptors with similar desensitization rates, suggesting that increased backbone to backbone H bonding expected in the absence of proline at position 180 would likely exert a great effect on desensitization. Single channel recordings indicated that for the α7-P180T receptor there was a significantly reduced closed time without any change in single channel conductance (as compared to wild-type). Kinetic simulations indicated that all changes observed for the mutant channel behaviour were reproduced by decreasing the rate of desensitization, and increasing the microscopic affinity to resting receptors. Molecular dynamics (MD) simulations on a homology model were used to provide insight into likely H bond interactions within the outer β-sheet that occur when the P180 residue is mutated. All mutations analysed increased about twofold the predicted number of H bonds between the residue at position 180 and the backbone of the β10 strand. Moreover, the α7-P180T and α7-P180S mutations also formed some intrastrand H bonds along the β9 strand, although H bonding of the OH groups of the threonine or serine side-chains was predicted to be infrequent. Our results indicate that rapid desensitization of the wild-type rat α7 nAChR is facilitated by the presence of the proline residue within the β9 strand.
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