Activation of Munc13-1 by Diacylglycerol (DAG)-Lactones.

Munc13-1 is a key protein necessary for vesicle fusion and neurotransmitter release in the brain. Diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane and activates it. The C1 domain of Munc13-1 and protein kinase C (PKC) are homologous in terms of sequence and structure. In order to identify small-molecule modulators of Munc13-1 targeting the C1 domain, we studied the effect of three DAG-lactones, (R,Z)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (JH-131e-153), (E)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (AJH-836), and (E)-(2-(hydroxymethyl)-4-(4-nitrobenzylidene)-5-oxotetrahydrofuran-2-yl)methyl 4-(dimethylamino)benzoate (130C037), on Munc13-1 activation using the ligand-induced membrane translocation assay. JH-131e-153 showed higher activation than AJH-836, and 130C037 was not able to activate Munc13-1. To understand the role of the ligand-binding site residues in the activation process, three alanine mutants were generated. For AJH-836, the order of activation was wild-type (WT) Munc13-1 > R592A > W588A > I590A. For JH-131e-153, the order of activation was WT > I590 ≈ R592A ≈ W588A. Overall, the Z isomer of DAG-lactones showed higher potency than the E isomer and Trp-588, Ile-590, and Arg-592 were important for its binding. When comparing the activation of Munc13-1 and PKC, the order of activation for JH-131e-153 was PKCα > Munc13-1 > PKCε and for AJH-836, the order of activation was PKCε > PKCα > Munc13-1. Molecular docking supported higher binding of JH-131e-153 than AJH-836 with the Munc13-1 C1 domain. Our results suggest that DAG-lactones have the potential to modulate neuronal processes via Munc13-1 and can be further developed for therapeutic intervention for neurodegenerative diseases.

Comparison of the ligand binding site of C1 domains: a molecular dynamics simulation study of the C1 domain-phorbol 13-acetate-membrane system.

C1 domains are lipid-binding structural units of about 50 residues. Typical C1 domains associate with the plasma membrane and bind to diacylglycerol/phorbol ester during the activation of the proteins containing these domains. Although the overall structure of the C1 domains are similar, there are differences in their primary sequence and in the orientation of the ligand/lipid binding residues. To gain structural insights into the ligand/lipid binding, we performed molecular docking of phorbol 13-acetate into the C1 domain and 1.0 µs molecular dynamics simulation on the C1 domain-ligand-lipid ternary system for PKCθ C1A, PKCδ C1B, PKCßII C1B, PKCθ C1B, Munc13-1 C1, and ßII-Chimaerin C1. We divided these C1 domains into three types based on the orientations of Gln-27 and Trp/Tyr-22. In type 1, Trp/Tyr-22 is outside and Gln-27 is inside the ligand binding pocket. In type 2, both Trp/Tyr-22 and Gln-27 are outside the ligand binding pocket, and in type 3, Trp/Tyr-22 is inside and Gln-27 is outside the pocket. The type 1 C1 domains showed higher ligand binding and higher membrane binding with a shorter distance between the C1 domain and the membrane than the type 2 and type 3. For ligand binding, Pro-11 plays a major role in the type 1 and 2, and Gly-23 in the type 1 and type 3 C1 domains. This study elucidates the role of Gln-27, Trp-22, Pro-11 and Gly-23 in ligand/lipid binding in typical C1 domains and bears significance in developing selective modulators of C1 domain-containing proteins.Communicated by Ramaswamy H. Sarma.

Differential Expression of Presynaptic Munc13-1 and Munc13-2 in Mouse Hippocampus Following Ethanol Drinking.

The Munc13 family of proteins is critically involved in synaptic vesicle priming and release in glutamatergic neurons in the brain. Munc13-1 binds to alcohol and, in Drosophila, modulates sedation sensitivity and self-administration. We examined the effect of alcohol consumption on the expression of Munc13-1 and Munc13-2, NMDA receptor subunits GluN1, GluN2A and GluN2B in the hippocampus-derived HT22 cells, hippocampal primary neuron culture, and wild-type and Munc13-1+/- male mouse hippocampus after ethanol consumption (Drinking in the Dark (DID) paradigm). In HT22 cells, Munc13-1 was upregulated following 25 mM ethanol treatment for 24 h. In the primary neuronal culture, however, the expression of both Munc13-1 and Munc13-2 increased after ethanol exposure. While Munc13-1 was upregulated in the hippocampus, Munc13-2 was downregulated following DID. This differential effect was found in the CA1 subfield of the hippocampus. Although Munc13-1+/- mice had approximately 50% Munc13-1 expression compared to wild-type, it was nonetheless significantly increased following DID. Munc13-1 and Munc13-2 were expressed in vesicular glutamate transporter1 (VGLUT1) immunoreactive neurons in the hippocampus, but ethanol did not alter the expression of VGLUT1. The NMDA receptor subunits, GluN1, GluN2A and GluN2B were upregulated in the hippocampal primary culture and in the CA1. Ethanol exerts a differential effect on the expression of Munc13-1 and Munc13-2 in the CA1 in male mice. Our study also found that ethanol's effect on Munc13 expression is dependent on the experimental paradigm, and both Munc13-1 and Munc13-2 could contribute to the ethanol-induced augmentation of glutamatergic neurotransmission.

Modulation of proteasome activity by curcumin and didemethylcurcumin.

Modulation of proteasome function by pharmacological interventions and molecular biology tools is an active area of research in cancer biology and neurodegenerative diseases. Curcumin (diferuloylmethane) is a naturally occurring polyphenol that affects multiple signaling pathways. Curcumin shows anti-inflammatory, antioxidant, anti-angiogenic, or anti-apoptotic properties. Recent research suggests that the therapeutic efficacy of curcumin may be due to its activity as a potent inhibitor of the proteasome. Using in vitro cell culture and molecular docking methods, here we show that both curcumin and its synthetic polyphenolic derivative (didemethylcurcumin, CUIII) modulated proteasome activity in a biphasic manner. Curcumin and CUIII increased proteasome activity at nanomolar concentrations, but inhibited proteasome activity at micromolar concentrations. Curcumin was more effective than CUIII in increasing relative proteasome activity at nanomolar concentrations. Also, curcumin was more effective than CUIII in inhibiting relative proteasome activity at micromolar concentrations. Docking simulations of curcumin and didemethylcurcumin binding to the 20S proteasome catalytic subunit estimated Kd values of 0.0054 µM and 1.3167 µM, respectively. These values correlate well with the results of the effectiveness of modulation by curcumin compared to CUIII. The small size of CUIII allows it to dock to the narrow cavity of the active site, but the binding interaction is not strong compared to curcumin. These results indicate that curcumin and its didemethyl derivative can be used to modulate proteasome activity and suggest that curcumin and its didemethyl derivative may be useful in treating two different disease classes: neurodegeneration and cancer.Communicated by Ramaswamy H. Sarma.

Molecular dynamics simulation studies on binding of activator and inhibitor to Munc13-1 C1 in the presence of membrane.

Munc13-1 is a presynaptic active zone protein that plays a critical role in priming the synaptic vesicle and releasing neurotransmitters in the brain. Munc13-1 acts as a scaffold and is activated when diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane. Our previous studies showed that bryostatin 1 activated the Munc13-1, but resveratrol inhibited the phorbol ester-induced Munc13-1 activity. To gain structural insights into the binding of the ligand into Munc13-1 C1 in the membrane, we conducted 1.0 µs molecular dynamics (MD) simulation on Munc13-1 C1-ligand-lipid ternary system using phorbol 13-acetate, bryostatin 1 and resveratrol as ligands. Munc13-1 C1 shows higher conformational stability and less mobility along membrane with phorbol 13-acetate and bryostatin 1 than with resveratrol. Bryostatin 1 and phorbol ester remained in the protein active site, but resveratrol moved out of Munc13-1 C1 during the MD simulation. While bryostatin 1-bound Munc13-1 C1 showed two different positioning in the membrane, phorbol 13-acetate and resveratrol-bound Munc13-1 C1 only showed one positioning. Phorbol 13-acetate formed hydrogen bond with Ala-574 and Gly-589. Bryostatin 1 had more hydrogen bonds with Trp-588 and Arg-592 than with other residues. Resveratrol formed hydrogen bond with Ile-590. This study suggests that different ligands control Munc13-1 C1's mobility and positioning in the membrane differently. Ligand also has a critical role in the interaction between Munc13-1 C1 and lipid membrane. Our results provide structural basis of the pharmacological activity of the ligands and highlight the importance of membrane in Munc13-1 activity.Communicated by Ramaswamy H. Sarma.

Comment on "Non-Markovian harmonic oscillator across a magnetic field and time-dependent force fields".

In a recent paper Das et al. [J. Chem. Phys. 147, 164102 (2017)JCPSA60021-960610.1063/1.4999408] proposed the Fokker-Planck equation (FPE) for the Brownian harmonic oscillator in the presence of a magnetic field and the non-Markovian thermal bath, respectively. This system has been studied very recently by Hidalgo-Gonzalez and Jiménez-Aquino [Phys. Rev. E 100, 062102 (2019)PREHBM2470-004510.1103/PhysRevE.100.062102] and the Fokker-Planck equation was derived using the characteristic function. It includes a few extra terms in the FPE and the authors conclude that their method is accurate compared to the calculation by Das et al. Then we reexamine our calculation and which is present in this comment. The revised calculation shows that both methods give the same result.

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1.

Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been implicated in the pathophysiology of several neurodegenerative diseases. Diacylglycerol and phorbol ester activate Munc13-1 by binding to its C1 domain. The objective of this study is to identify the structural determinants of ligand binding activity of the Munc13-1 C1 domain. Molecular docking suggested that residues Trp-588, Ile-590, and Arg-592 of Munc13-1 are involved in ligand interactions. To elucidate the role of these three residues in ligand binding, we generated W588A, I590A, and R592A mutants in full-length Munc13-1, expressed them as GFP-tagged proteins in HT22 cells, and measured their ligand-induced membrane translocation by confocal microscopy and immunoblotting. The extent of 1,2-dioctanoyl-sn-glycerol (DOG)- and phorbol ester-induced membrane translocation decreased in the following order: wild type > I590A > W588A > R592A and wild type > W588A > I590A > R592A, respectively. To understand the effect of the mutations on ligand binding, we also measured the DOG binding affinity of the isolated wild-type C1 domain and its mutants in membrane-mimicking micelles using nuclear magnetic resonance methods. The DOG binding affinity decreased in the following order: wild type > I590A > R592A. No binding was detected for W588A with DOG in micelles. This study shows that Trp-588, Ile-590, and Arg-592 are essential determinants for the activity of Munc13-1 and the effects of the three residues on the activity are ligand-dependent. This study bears significance for the development of selective modulators of Munc13-1.

Unified approach to stochastic thermodynamics: Application to a quantum heat engine.

In the present study we have developed an alternative formulation for the quantum stochastic thermodynamics based on the c-number Langevin equation for the system-reservoir model. This is analogous to the classical one. Here we have considered both Markovian and non-Markovian dynamics (NMD). Consideration of the NMD is an important issue at the current state of the stochastic thermodynamics. Applying the present formalism, we have carried out a comparative study on the heat absorbed and the change of entropy with time for a linear quantum system and its classical analog for both Markovian and NMD. Here the strength of the thermal noise and its correlation time for the respective cases are the leading quantities to explain any distinguishable feature which may appear with the equilibration kinetics. For another application, we have proposed a formulation with classical look for a quantum stochastic heat engine. Using it we have presented a comparative study on the efficiency and its value at maximum power for a quantum stochastic heat engine and its classical analog. The engines are Carnot like which are coupled with their respective Markovian thermal baths. Here also the noise strength as well as the diffusion constant are the leading quantities to explain any noticeable feature.

Repurposing of Drugs-The Ketamine Story.

An intranasal formulation of esketamine, the S enantiomer of ketamine, in conjunction with an oral antidepressant, has been approved by the FDA for treating treatment-resistant major depressive disorder (TRD) in 2019, almost 50 years after it was approved as an intravenous anesthetic. In contrast to traditional antidepressants, ketamine shows a rapid (within 2 h) and sustained (∼7 days) antidepressant effect and has significant positive effects on antisuicidal ideation. Ketamine's antidepressant mechanism is predominantly mediated by the N-methyl-d-aspartate receptor (NMDA) receptor, although NMDA-independent mechanisms are not ruled out. At the neurocircuitry level, ketamine affects the brain's reward and mood circuitry located in the corticomesolimbic structures involving the hippocampus, nucleus accumbens, and prefrontal cortex. Repurposing of ketamine for treating TRD provided a new understanding of the pathophysiology of depression, a paradigm shift from monoamine to glutamatergic neurotransmission, thus making it a unique tool to investigate the brain and its complex neurocircuitries.

Effect of ethanol on Munc13-1 C1 in Membrane: A Molecular Dynamics Simulation Study.

BACKGROUND: EtOH has a significant effect on synaptic plasticity. Munc13-1 is an essential presynaptic active zone protein involved in priming the synaptic vesicle and releasing neurotransmitter in the brain. It is a peripheral membrane protein and binds to the activator, diacylglycerol (DAG)/phorbol ester at its membrane-targeting C1 domain. Our previous studies identified Glu-582 of C1 domain as the alcohol-binding residue (Das, J. et al, J. Neurochem., 126, 715-726, 2013). METHODS: Here, we describe a 250 ns molecular dynamics (MD) simulation study on the interaction of EtOH and the activator-bound Munc13-1 C1 in the presence of varying concentrations of phosphatidylserine (PS). RESULTS: In this study, Munc13-1 C1 shows higher conformational stability in EtOH than in water. It forms fewer hydrogen bonds with phorbol 13-acetate in the presence of EtOH than in water. EtOH also affected the interaction between the protein and the membrane and between the activator and the membrane. Similar studies in a E582A mutant suggest that these effects of EtOH are mostly mediated through Glu-582. CONCLUSIONS: EtOH forms hydrogen bonds with Glu-582. While occupancy of the EtOH molecules at the vicinity (4Å) of Glu-582 is 34.4%, the occupancy in the E582A mutant is 26.5% of the simulation time. In addition, the amount of PS in the membrane influences the conformational stability of the C1 domain and interactions in the ternary complex. This study is important in providing the structural basis of EtOH's effects on synaptic plasticity.

MUNC13-1 heterozygosity does not alter voluntary ethanol consumption or sensitivity in mice.

The role of the munc13-1 presynaptic protein in alcohol-related behaviors has been little-studied, despite being a known site of action for ethanol binding. Munc13-1 is an active zone protein that plays a vital role in vesicle maturation and the release of neurotransmitters in excitatory neurons. Ethanol binds munc13-1, which decreases its functionality. In Drosophila, loss of the homologous protein Dunc13 is associated with an increase in ethanol preference, and is associated with a resistance to sedation following ethanol exposure. The current study assessed the effects of munc13-1 heterozygosity on ethanol sensitivity and consumption in mice, as well as on learning and anxiety-like behaviors, which can influence alcohol intake. Wild-type and mutant mice underwent 6 cycles of drinking-in-the-dark (DID) as well as rotarod testing following ethanol injection, to probe for differences in ethanol consumption and sensitivity, respectively. We did not detect genotype-based differences in our measures of anxiety, spatial learning, ethanol consumption, or ethanol sensitivity. However, heterozygotes showed increased use of a spatial navigation strategy in a dual-solution water maze, as opposed to a stimulus-response strategy. To summarize, although reduction of Dunc13 in flies produces clear effects on ethanol consumption and sensitivity, heterozygosity for munc13-1 does not, potentially due to compensatory adaptation by other munc-13 isoforms.

SNARE Complex-Associated Proteins and Alcohol.

Alcohol addiction causes major health problems throughout the world, causing numerous deaths and incurring a huge economic burden to society. To develop an intervention for alcohol addiction, it is necessary to identify molecular target(s) of alcohol and associated molecular mechanisms of alcohol action. The functions of many central and peripheral synapses are impacted by low concentrations of ethanol (EtOH). While the postsynaptic targets and mechanisms are studied extensively, there are limited studies on the presynaptic targets and mechanisms. This article is an endeavor in this direction, focusing on the effect of EtOH on the presynaptic proteins associated with the neurotransmitter release machinery. Studies on the effects of EtOH at the levels of gene, protein, and behavior are highlighted in this article.

Munc13 Is a Molecular Target of Bryostatin 1.

Bryostatin 1 is a natural macrolide shown to improve neuronal connections and enhance memory in mice. Its mechanism of action is largely attributed to the modulation of novel and conventional protein kinase Cs (PKCs) by binding to their regulatory C1 domains. Munc13-1 is a C1 domain-containing protein that shares common endogenous and exogenous activators with novel and conventional PKC subtypes. Given the essential role of Munc13-1 in the priming of synaptic vesicles and neuronal transmission overall, we explored the potential interaction between bryostatin 1 and Munc13-1. Our results indicate that in vitro bryostatin 1 binds to both the isolated C1 domain of Munc13-1 ( Ki = 8.07 ± 0.90 nM) and the full-length Munc13-1 protein ( Ki = 0.45 ± 0.04 nM). Furthermore, confocal microscopy and immunoblot analysis demonstrated that in intact HT22 cells bryostatin 1 mimics the actions of phorbol esters, a previously established class of Munc13-1 activators, and induces plasma membrane translocation of Munc13-1, a hallmark of its activation. Consistently, bryostatin 1 had no effect on the Munc13-1H567K construct that is insensitive to phorbol esters. Effects of bryostatin 1 on the other Munc13 family members, ubMunc13-2 and bMunc13-2, resembled those of Munc13-1 for translocation. Lastly, we observed an increased level of expression of Munc13-1 following a 24 h incubation with bryostatin 1 in both HT22 and primary mouse hippocampal cells. This study characterizes Munc13-1 as a molecular target of bryostatin 1. Considering the crucial role of Munc13-1 in neuronal function, these findings provide strong support for the potential role of Munc13s in the actions of bryostatin 1.

Identification of alcohol-binding site(s) in proteins using diazirine-based photoaffinity labeling and mass spectrometry.

Defining molecular targets of alcohol and understanding the molecular mechanism of alcohol actions are necessary to develop effective therapeutics for alcohol use disorder (AUD). Here, we describe a detailed protocol for identifying alcohol-binding site(s) in proteins using diazirine-based azialcohol as photoaffinity labeling agents. Upon photoirradiation, azialcohol photoincorporates into alcohol-binding proteins. The stoichiometry and site of azialcohol photoincorporation can be determined using high-resolution mass spectrometry. Identification of the alcohol-binding residues in protein followed by measuring the biological significance of these residues in regulating alcohol action are important steps in characterizing the molecular targets of alcohol.

Autonomous stochastic resonance driven by colored noise.

A one-dimensional linear autonomous system coupled to a generic stationary nonequilibrium fluctuating bath can exhibit resonant response when its damped oscillation period matches some characteristic bath's relaxation time. This condition justifies invoking the stochastic resonance paradigm, even if it can be achieved more easily by tuning the system to the bath and not vice versa, as is usually the case. The simple nature of the mechanism numerically investigated here suggests a number of interesting applications for instance in the context of (1) energy harvesting from random ambient vibrations, (2) activated barrier crossing through a saddle point, or (3) an unstable limit cycle.

Ethanol Regulates Presynaptic Activity and Sedation through Presynaptic Unc13 Proteins in Drosophila.

Ethanol has robust effects on presynaptic activity in many neurons, however, it is not yet clear how this drug acts within this compartment to change neural activity, nor the significance of this change on behavior and physiology in vivo. One possible presynaptic effector for ethanol is the Munc13-1 protein. Herein, we show that ethanol binding to the rat Munc13-1 C1 domain, at concentrations consistent with binge exposure, reduces diacylglycerol (DAG) binding. The inhibition of DAG binding is predicted to reduce the activity of Munc13-1 and presynaptic release. In Drosophila, we show that sedating concentrations of ethanol significantly reduce synaptic vesicle release in olfactory sensory neurons (OSNs), while having no significant impact on membrane depolarization and Ca2+ influx into the presynaptic compartment. These data indicate that ethanol targets the active zone in reducing synaptic vesicle exocytosis. Drosophila, haploinsufficent for the Munc13-1 ortholog Dunc13, are more resistant to the effect of ethanol on presynaptic inhibition. Genetically reducing the activity of Dunc13 through mutation or expression of RNAi transgenes also leads to a significant resistance to the sedative effects of ethanol. The neuronal expression of Munc13-1 in heterozygotes for a Dunc13 loss-of-function mutation can largely rescue the ethanol sedation resistance phenotype, indicating a conservation of function between Munc13-1 and Dunc13 in ethanol sedation. Hence, reducing Dunc13 activity leads to naïve physiological and behavioral resistance to sedating concentrations of ethanol. We propose that reducing Dunc13 activity, genetically or pharmacologically by ethanol binding to the C1 domain of Munc13-1/Dunc13, promotes a homeostatic response that leads to ethanol tolerance.

Structural determinants of phorbol ester binding activity of the C1a and C1b domains of protein kinase C theta.

The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro9 of the C1a domain of PKCθ to the corresponding Lys9 found in C1b restored in vitro binding activity for [3H]phorbol 12,13-dibutyrate to 3.6 nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys9 to Pro9 only diminished binding affinity to 11.7 nM, compared to 254 nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100 nM PMA) the translocation to the plasma membrane. We conclude that the Pro168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys240) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction.

Case-based studies in teaching medicinal chemistry in PharmD curriculum: Perspectives of students, faculty, and pharmacists from academia.

BACKGROUND AND PURPOSE: Pharmacy practice has evolved tremendously over the years to meet the demands of the growing healthcare system. Foundational sciences like, medicinal chemistry can enhance the critical-thinking and therapeutic decision-making skills of today's professional pharmacists. The importance of medicinal chemistry for the doctor of pharmacy (PharmD) curriculum has been discussed from the perspectives of medicinal chemistry and practicing clinical faculty whose focused practices vary from infectious diseases to geriatrics. EDUCATIONAL ACTIVITY AND SETTING: An Institutional Review Board (IRB)-approved perception survey and a year-end course evaluation were given to the second and third professional year students. FINDINGS: Eighty-eight percent of the participating second-year students and 92% of the participating third-year students thought that the introduction of case studies in the medicinal chemistry curriculum enhanced their learning and appreciation for the subject. DISCUSSION AND SUMMARY: The Pharmacy Curriculum Outcomes Assessment (PCOA) exams, given at the University of Houston College of Pharmacy during the years of 2013-2015, were briefly discussed. Since the requirement to administer the PCOA went into effect in early 2016, the authors felt that not enough time existed to establish meaningful controls to conduct a correlation study with the student perspective survey results obtained and PCOA data provided in 2015. This study, therefore, highlights the importance of integrated approaches to pharmacy teaching at the University of Houston.

Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation.

Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the role of this tryptophan, we generated W22A, W22K, W22D, W22Y, and W22F substitutions in the full-length Munc13-1, expressed the GFP-tagged constructs in Neuro-2a cells, and measured their membrane translocation in response to phorbol ester treatment by imaging of the live cells using confocal microscopy. The extent of membrane translocation followed the order, wild-type > W22K > W22F > W22Y > W22A > W22D. The phorbol ester binding affinity of the wild-type Munc13-1C1 domain and its mutants was phosphatidylserine (PS)-dependent following the order, wild-type > W22K > W22A ≫ W22D in both 20% and 100% PS. Phorbol ester affinity was higher for Munc13-1 than the C1 domain. While Munc13-1 translocated to the plasma membrane, the C1 domain translocated to internal membranes in response to phorbol ester. Molecular dynamics (80 ns) studies reveal that Trp-22 is relatively less flexible than the homologous Trp-22 of PKCδ and PKCθ. Results are discussed in terms of the overall negative charge state of the Munc13-1C1 domain and its possible interaction with the PS-rich plasma membrane. This study shows that Trp-588 is an important structural element for ligand binding and membrane translocation in Munc13-1.

Fokker-Planck equation for the non-Markovian Brownian motion in the presence of a magnetic field.

In the present study, we have proposed the Fokker-Planck equation in a simple way for a Langevin equation of motion having ordinary derivative (OD), the Gaussian random force and a generalized frictional memory kernel. The equation may be associated with or without conservative force field from harmonic potential. We extend this method for a charged Brownian particle in the presence of a magnetic field. Thus, the present method is applicable for a Langevin equation of motion with OD, the Gaussian colored thermal noise and any kind of linear force field that may be conservative or not. It is also simple to apply this method for the colored Gaussian noise that is not related to the damping strength.