Competitive CatSper Activators of Progesterone from Rhynchosia volubilis

The root Rhynchosia volubilis was widely used for contraception in folk medicine, although its molecular mechanism on antifertility has not yet been revealed. In human sperm, it was reported that the cation channel of sperm, an indispensable cation channel for the fertilization process, could be regulated by various steroid-like compounds in plants. Interestingly, these nonphysiological ligands would also disturb the activation of the cation channel of sperm induced by progesterone. Therefore, this study aimed to explore whether the compounds in R. volubilis affect the physiological regulation of the cation channel of sperm. The bioguided isolation of the whole herb of R. volubilis has resulted in the novel discovery of five new prenylated isoflavonoids, rhynchones A – E (1 – 5), a new natural product, 5′-O-methylphaseolinisoflavan (6) (1H and 13C NMR data, Supporting Information), together with twelve known compounds (7 – 18). Their structures were established by extensive spectroscopic analyses and drawing a comparison with literature data, while their absolute configurations were determined by electronic circular dichroism calculations. The experiments of intracellular Ca2+ signals and patch clamping recordings showed that rhynchone A (1) significantly reduced cation channel of sperm activation by competing with progesterone. In conclusion, our findings indicat that rhynchone A might act as a contraceptive compound by impairing the activation of the cation channel of sperm and thus prevent fertilization.

Injection with Toxoplasma gondii protein affects neuron health and survival

Toxoplasma gondii is an intracellular parasite that causes a long-term latent infection of neurons. Using a custom MATLAB-based mapping program in combination with a mouse model that allows us to permanently mark neurons injected with parasite proteins, we found that Toxoplasma-injected neurons (TINs) are heterogeneously distributed in the brain, primarily localizing to the cortex followed by the striatum. In addition, we determined that cortical TINs are commonly (>50%) excitatory neurons (FoxP2+) and that striatal TINs are often (>65%) medium spiny neurons (MSNs) (FoxP2+). By performing single neuron patch-clamping on striatal TINs and neighboring uninfected MSNs, we discovered that TINs have highly aberrant electrophysiology. As approximately 90% of TINs will die by 8 weeks post-infection, this abnormal physiology suggests that injection with Toxoplasma protein— either directly or indirectly— affects neuronal health and survival. Collectively, these data offer the first insights into which neurons interact with Toxoplasma and how these interactions alter neuron physiology in vivo.

7. Following biochemistry within the cell

This chapter presents key advances in the study of individual molecules within cells. When one applies this individualistic methodology to biochemicals, one enters the realms of single-molecule biophysics. There are of course formidable technical challenges associated with single-molecule studies, not least of which is the signal-to-noise problem. The chapter discusses patch clamping, nanoscopes, and optical tweezers. Patch clamping provided insights into the protein machinery that controls the flow of ions in and out of cells. The chapter also examines cytokinesis, cytoskeletons, and motor proteins, and the use of Green Fluorescent Protein (GFP).

Deep learning-based real-time detection of neurons in brain slices for in vitro physiology

A common electrophysiology technique used in neuroscience is patch clamp: a method in which a glass pipette electrode facilitates single cell electrical recordings from neurons. Typically, patch clamp is done manually in which an electrophysiologist views a brain slice under a microscope, visually selects a neuron to patch, and moves the pipette into close proximity to the cell to break through and seal its membrane. While recent advances in the field of patch clamping have enabled partial automation, the task of detecting a healthy neuronal soma in acute brain tissue slices is still a critical step that is commonly done manually, often presenting challenges for novices in electrophysiology. To overcome this obstacle and progress towards full automation of patch clamp, we combined the differential interference microscopy optical technique with an object detection-based convolutional neural network (CNN) to detect healthy neurons in acute slice. Utilizing the YOLOv3 convolutional neural network architecture, we achieved a 98% reduction in training times to 18 min, compared to previously published attempts. We also compared networks trained on unaltered and enhanced images, achieving up to 77% and 72% mean average precision, respectively. This novel, deep learning-based method accomplishes automated neuronal detection in brain slice at 18 frames per second with a small data set of 1138 annotated neurons, rapid training time, and high precision. Lastly, we verified the health of the identified neurons with a patch clamp experiment where the average access resistance was 29.25 M$$\Omega$$ Ω (n = 9). The addition of this technology during live-cell imaging for patch clamp experiments can not only improve manual patch clamping by reducing the neuroscience expertise required to select healthy cells, but also help achieve full automation of patch clamping by nominating cells without human assistance.

Toxoplasma gondii injected neurons localize to the cortex and striatum and have altered firing

Toxoplasma gondii is an intracellular parasite that causes a long-term latent infection of neurons. Using a custom MATLAB-based mapping program in combination with a mouse model that allows us to permanently mark neurons injected with parasite proteins, we found that Toxoplasma-injected neurons (TINs) are heterogeneously distributed in the brain, primarily localizing to the cortex followed by the striatum. Using immunofluorescence co-localization assays, we determined that cortical TINs are commonly (>50%) excitatory neurons (FoxP2+) and that striatal TINs are often (>65%) medium spiny neurons (MSNs) (FoxP2+). As MSNs have highly characterized electrophysiology, we used ex vivo slices from infected mice to perform single neuron patch-clamping on striatal TINs and neighboring uninfected MSNs (bystander MSNs). These studies demonstrated that TINs have highly abnormal electrophysiology, while the electrophysiology of bystander MSNs was akin to that of MSNs from uninfected mice. Collectively, these data offer new neuroanatomic and electrophysiologic insights into CNS toxoplasmosis.

Electrophysiological evidence of synergistic auxin transport by interacting Arabidopsis ABCB4 and PIN2 proteins

To understand why both ATP-binding cassette B (ABCB) and PIN-FORMED (PIN) proteins are required for polar auxin transport through tissues, even though only the latter is polarly localized, we biophysically studied their transport characteristics separately and together by whole-cell patch clamping. ABCB4 and PIN2 from Arabidopsis thaliana expressed in human embryonic kidney cells displayed electrogenic activity when CsCl-based electrolytes were used. Current-voltage (I-V) analysis of the activities and modeling the effects of adding the auxin anion (IAA−) as a potential substrate with the Goldman-Hodgkin-Katz equation, demonstrated that ABCB4 and PIN2 were 9-fold and 10-fold more selective for IAA− than Cl−, respectively. Thus, these proteins directly transport IAA−, which was not unequivocally established by previous auxin retention assays. Co-expression of ABCB4 and PIN2 produced an especially significant result. Co-expression synergistically doubled the selectivity for IAA−. An area of two-fold higher selectivity for IAA− that this result indicates will occur in cells with asymmetric PIN2 and symmetric ABCB4 matches what early models found to be necessary to create observed levels of polar auxin transport through tissues. Thus, the requirement for two different proteins appears to be explained by a synergistic effect on selectivity. More substrate details and important pharmacological results are reported.

Proton receptors regulate synapse-specific reconsolidation in the amygdala

During retrieval, aversive memories become labile during a period known as the reconsolidation window. When an extinction procedure is performed within the reconsolidation window, the original aversive memory can be replaced by one that is less traumatic. Our recent studies revealed that acidosis via inhalation of carbon dioxide (CO2) during retrieval enhances memory lability. However, the effects of CO2 inhalation on the central nervous system can be extensive, and there is a lack of prior evidence suggesting that the effects of CO2 are selective to a reactivated memory. The specific effects of CO2 depend on acid-sensing ion channels (ASICs), proton receptors that are involved in synaptic transmission and plasticity in the amygdala. Our previous patch-clamping data suggests that CO2 inhalation during retrieval increases activities of neurons in the amygdala that involve in the memory trace. In addition, CO2 inhalation during retrieval increases exchanges from Ca2+-impermeable to Ca2+-permeable AMPA receptors. Thus, we hypothesize that CO2 selectively potentiates memory lability in mice when inhaled during retrieval of aversive memory. In addition, CO2 inhalation alters memory lability via synaptic plasticity at selectively targeted synapses. Alterations in spine morphology after CO2 and retrieval with a specific stimulus indicates that CO2 selectively enhances synaptic plasticity. Overall, our results suggest that inhaling CO2 during the retrieval event increases the lability of an aversive memory through a synapse-specific reconsolidation process.

Abstract 15438: Comprehensive Structural, Molecular, and Electrophysiological Maturation of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes to Serve as a Platform to Model Atrial Fibrillation

Introduction: Increasingly, human induced pluripotent stem cells (hiPSC) faithfully recapitulate human models of arrhythmias. However, enhancing hiPSC-derived atrial cardiomyocyte (aCM) maturity is vital as modeling mature CMs will provide insights into cellular mechanisms of atrial fibrillation (AF) and signaling pathways critical to atrial development Hypothesis: Combinatorial conditioning of hiPSC-aCMs with biochemical cues (T3, IGF-1, dexamethasone; TID), fatty acids (FA; oleic/palmitic acid), and acute electrical stimulation (ES) at increasing intensity over 45 days comprehensively enhances structural, molecular, and electrophysiological (EP) maturity of hiPSC-aCMs Methods: HiPSCs generated from patient specific peripheral blood mononuclear cells were differentiated into aCMs using retinoic acid and glucose starvation. Maturity of atrial iPSC-CMs was enhanced using TID, FA, and acute ES for the final 4 weeks of culture. Structural (immunofluorescence; transmission EM), molecular (qPCR; RNAseq), and EP (patch clamping; multielectrode array; high throughput automated patch clamping) maturity is assessed and compared to untreated hiPSC-aCMs and adult human aCMs harvested from the same patient (optimal maturity) Results: We showed improved hiPSC-aCM structural maturity with TID, FA, and ES ( Fig. 1A ). EP maturity also displayed more hyperpolarized resting membrane potential (RMP; Fig. 1B ), and improved upstroke velocity, action potential duration (APD), and amplitude (not shown). Expression of ion channels, and calcium handling and structural proteins is significantly improved ( Fig. 1C ) Conclusions: Combinatorial conditioning with TID, FA, and ES markedly improved structural, molecular, and EP maturity of hiPSC-aCMs. Our findings will serve as a platform to model AF, elucidate underlying cellular mechanisms, and identify novel therapeutic targets for a personalized, mechanism based approach to treat this common condition

Abstract 14836: Dual Dysfunction of Kir2.1 at the Sarcolemma and the Sarcoplasmic Reticulum Underlies Arrhythmogenesis in a Mouse Model of the Andersen-Tawil Syndrome Type 1

Introduction: Andersen-Tawil syndrome type 1 (ATS1) is associated with fatal cardiac arrhythmias. However, the underlying mechanisms are poorly understood. Hypothesis: Cardiac-specific expression of trafficking deficient Kir2.1 channels in mice in-vivo recapitulates the cardiac electrical phenotype of ATS1 and investigate the underlying mechanisms. Methods: We generated a new mouse model of ATS1 by a single i.v. injection of cardiac specific adeno-associated viral (AAV) transduction with Kir2.1 Δ314-315 , which recapitulated the ATS1 ECG phenotype without modifying ventricular function. The animal and cellular, structural and functional analyses were carried out by ECG, intracardiac stimulation, patch-clamping, membrane fractionation, western blot, immunolocalization and live calcium imaging. Results: AAV-Kir2.1 Δ314-315 mice were significantly more sensitive to flecainide than control, increasing the PR and QRS intervals over time. Kir2.1 Δ314-315 mice had increased vulnerability to cardiac fibrillation. Patch-clamping in ventricular cardiomyocytes from Kir2.1 Δ314-315 mice demonstrated significantly reduced I K1 and I Na , depolarized resting membrane potential and prolonged action potential. Immunolocalization in control mice revealed two bands of Kir2.1 staining, one colocalizing with Na V 1.5 and AP1 near the Z disk, the other near the H zone. Membrane fractionation and western blot experiments demonstrated two distinct levels of Kir2.1 protein expression, one at the sarcolemmal fraction together with Na V 1.5 and the other at the sarcoplasmi creticulum (SR). Kir2.1 Δ314-315 cardiomyocytes showed disruption of the Kir2.1-Nav1.5 channelosome at the sarcolemma, indicating dysfunctional trafficking o fboth channels. In addition, the SR Kir2.1 was disrupted and calcium transient dynamics were altered, resulting in frequent abnormal spontaneous calcium release events, and revealing an excitation-contraction coupling defect. Conclusions: Cardiac-specific AAV transduction with Kir2.1 Δ314-315 in mice recapitulates the ATS1 phenotype by disrupting localization and function of Kir2.1at the SR, and the Kir2.1-Na V 1.5 channelosome at the sarcolemma, revealing anovel dual mechanism of arrhythmogenesis.

Oxidative stress induced by the anti-cancer agents, plumbagin, and atovaquone, inhibits ion transport through Na+/K+-ATPase

Oxidative stress inhibits Na+/K+-ATPase (NKA), the ion channel that maintains membrane potential. Here, we investigate the role of oxidative stress-mediated by plumbagin and atovaquone in the inhibition of NKA activity. We confirm that plumbagin and atovaquone inhibit the proliferation of three human (OVCAR-3, SKOV-3, and TYKNu) and one mouse (ID8) ovarian cancer cell lines. The oxygen radical scavenger, N-acetylcysteine (NAC), attenuates the chemotoxicity of plumbagin and atovaquone. Whole-cell patch clamping demonstrates that plumbagin and atovaquone inhibit outward and the inward current flowing through NKA in SKOV-3 and OVCAR-3. Although both drugs decrease cellular ATP; providing exogenous ATP (5 mM) in the pipet solution used during patch clamping did not recover NKA activity in the plumbagin or atovaquone treated SKOV-3 and OVCAR-3 cells. However, pretreatment of the cells with NAC completely abrogated the NKA inhibitory activity of plumbagin and atovaquone. Exposure of the SKOV-3 cells to either drug significantly decreases the expression of NKA. We conclude that oxidative stress caused by plumbagin and atovaquone degrades NKA, resulting in the inability to maintain ion transport. Therefore, when evaluating compounds that induce oxidative stress, it is important to consider the contribution of NKA inhibition to their cytotoxic effects on tumor cells.