Initial Coupling of Binding to Gating Mediated by Conserved Residues in the Muscle Nicotinic Receptor

We examined functional consequences of intrasubunit contacts in the nicotinic receptor α subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues αK145, αD200, and αY190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations αD200N and αK145Q impair channel gating to the same extent as either single mutation, while αK145E counteracts the impaired gating due to αD200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, αK145 and αD200 form a salt bridge associated with the closed state of the channel. When ACh binds, αY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches αK145, which in turn loosens its contact with αD200. The positional changes of αK145 and αD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of β-strand 7, which harbors αK145 and is part of the signature Cys-loop, and the second is of β-strand 10, which harbors αD200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel

Mechanism of calcium potentiation of the α7 nicotinic acetylcholine receptor

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.Fil: Natarajan, Kathiresan. Mayo Clinic Cancer Center; Estados UnidosFil: Mukhtasimova, Nuriya. Mayo Clinic Cancer Center; Estados UnidosFil: Corradi, Jeremias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Lasala, Matías Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Sine, Steven M.. Mayo Clinic Cancer Center; Estados Unido

PROGRAM PELATIHAN DETEKSI DINI DAN STIMULASI TUMBUH KEMBANG PADA ORANG TUA SERTA GURU ANAK USIA 3-6 TAHUN DI TK DHARMA WANITA DUKUH 2 KECAMATAN NGADILUWIH

Kegiatan Pengabdian kepada Masyarakat (PKM) berupa “Program Pelatihan Deteksi Dini Dan Stimulasi Tumbuh Kembang Pada Orang tua serta guru Anak Usia 3-6 Tahun Di TK Dharma Wanita Dukuh 2 Kecamatan Ngadiluwihâ€. Program ini dilatarbelakangi indikator SDG’s 4.2.1 yaitu anak usia dibawah 5 tahun yang berkembang dengan baik (on the track) dalam bidang Kesehatan, pembelajaran dan psikososial (BPS, 2018). Populasi anak Indonesia mencapai 31,56 % dari total seluruh penduduk, yaitu 84.4 juta anak (Kemen PPPA, 2020). Terdapat lebih dari 200 juta anak balita di seluruh dunia diperkirakan mengalami gangguan perkembangan kognitif dan sosial emosional (Zhang J et al, 2018). Anak menjadi modal kemajuan bangsa yang perlu mendapat jaminan pertumbuhan dan perkembangan yang optimal (BPS, 2018). Setiap orang tua mengharapkan tumbuh kembang optimal bagi anak mereka, sayangnya hal ini tidak diiringi pengetahuan dan kemampuan yang optimal dan deteksi dini serta stimulasi tumbuh kembang anak. Penelitian menunjukkan adanya hubungan antara pengetahuan ibu terhadap perkembangan motorik anak usia balita di TK Dharma Wanita Desa Ringinsari Kediri (Eko Sri, 2022).  Tujuan kegiatan PKM yaitu 1) menjadi program percontohan dalam optimalisasi tumbuh kembang anak usia 3-6 tahun; 2) meningkatkan pengetahuan dan kemampuan orang tua dan guru yang memiliki/mengajar anak usia 3-6 tahun untuk dapat melakukan deteksi dini dan simulasi tumbuh kembang secara mandiri dan berkesinambungan. Target luaran yang ingin dicapai yaitu publikasi artikel pada jurnal ber ISSN (luaran wajib) serta modul aplikatif. Metode yang digunakan berupa penyuluhan, demo, pelatihan dan monitoring pasca kegiatan. Kegiatan PKM berlangsung selama 6 bulan (Agustus sampai Januari 2022)

Clinical cases of microscopic colitis: Diagnosis and treatment issues. Case report

Currently, there is an increase in the incidence of microscopic colitis. There are difficulties in diagnosing this disease due to the variability of histological signs, variability of morphological changes in the mucous membrane of the colon in different parts of the colon, and the combination in one patient of not only various forms of microscopic colitis, but also other intestinal diseases. The article describes the differential diagnosis, an example of its staging and successful treatment of various forms of microscopic colitis with budesonide (two clinical cases presented)

High Speed Two-Photon Imaging of Calcium Dynamics in Dendritic Spines: Consequences for Spine Calcium Kinetics and Buffer Capacity

Rapid calcium concentration changes in postsynaptic structures are crucial for synaptic plasticity. Thus far, the determinants of postsynaptic calcium dynamics have been studied predominantly based on the decay kinetics of calcium transients. Calcium rise times in spines in response to single action potentials (AP) are almost never measured due to technical limitations, but they could be crucial for synaptic plasticity. With high-speed, precisely-targeted, two-photon point imaging we measured both calcium rise and decay kinetics in spines and secondary dendrites in neocortical pyramidal neurons. We found that both rise and decay kinetics of changes in calcium-indicator fluorescence are about twice as fast in spines. During AP trains, spine calcium changes follow each AP, but not in dendrites. Apart from the higher surface-to-volume ratio (SVR), we observed that neocortical dendritic spines have a markedly smaller endogenous buffer capacity with respect to their parental dendrites. Calcium influx time course and calcium extrusion rate were both in the same range for spines and dendrites when fitted with a dynamic multi-compartment model that included calcium binding kinetics and diffusion. In a subsequent analysis we used this model to investigate which parameters are critical determinants in spine calcium dynamics. The model confirmed the experimental findings: a higher SVR is not sufficient by itself to explain the faster rise time kinetics in spines, but only when paired with a lower buffer capacity in spines. Simulations at zero calcium-dye conditions show that calmodulin is more efficiently activated in spines, which indicates that spine morphology and buffering conditions in neocortical spines favor synaptic plasticity

Synapse Geometry and Receptor Dynamics Modulate Synaptic Strength

Synaptic transmission relies on several processes, such as the location of a released vesicle, the number and type of receptors, trafficking between the postsynaptic density (PSD) and extrasynaptic compartment, as well as the synapse organization. To study the impact of these parameters on excitatory synaptic transmission, we present a computational model for the fast AMPA-receptor mediated synaptic current. We show that in addition to the vesicular release probability, due to variations in their release locations and the AMPAR distribution, the postsynaptic current amplitude has a large variance, making a synapse an intrinsic unreliable device. We use our model to examine our experimental data recorded from CA1 mice hippocampal slices to study the differences between mEPSC and evoked EPSC variance. The synaptic current but not the coefficient of variation is maximal when the active zone where vesicles are released is apposed to the PSD. Moreover, we find that for certain type of synapses, receptor trafficking can affect the magnitude of synaptic depression. Finally, we demonstrate that perisynaptic microdomains located outside the PSD impacts synaptic transmission by regulating the number of desensitized receptors and their trafficking to the PSD. We conclude that geometrical modifications, reorganization of the PSD or perisynaptic microdomains modulate synaptic strength, as the mechanisms underlying long-term plasticity

Quiescence and γH2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase

Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21Waf1/Cip1 and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. Conclusion: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies

Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafficking by JNK

Jun N-terminal kinases (JNKs) are implicated in various neuropathological conditions. However, physiological roles for JNKs in neurons remain largely unknown, despite the high expression level of JNKs in brain. Here, using bioinformatic and biochemical approaches, we identify the AMPA receptor GluR2L and GluR4 subunits as novel physiological JNK substrates in vitro, in heterologous cells and in neurons. Consistent with this finding, GluR2L and GluR4 associate with specific JNK signaling components in the brain. Moreover, the modulation of the novel JNK sites in GluR2L and GluR4 is dynamic and bi-directional, such that phosphorylation and de-phosphorylation are triggered within minutes following decreases and increases in neuronal activity, respectively. Using live-imaging techniques to address the functional consequence of these activity-dependent changes we demonstrate that the novel JNK site in GluR2L controls reinsertion of internalized GluR2L back to the cell surface following NMDA treatment, without affecting basal GluR2L trafficking. Taken together, our results demonstrate that JNK directly regulates AMPA-R trafficking following changes in neuronal activity in a rapid and bi-directional manner

The Biochemistry, Ultrastructure, and Subunit Assembly Mechanism of AMPA Receptors

The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity