Mechanism Of Constriction Research Articles

Mechanism of selectivity filter constriction in potassium channel: Insights from high-throughput steered molecular dynamics simulations

Potassium channels are essential for regulating cellular excitability by controlling K+ ion flow. In voltage-gated potassium (Kv) channels, C-type inactivation modulates action potentials and holds significant physiological and clinical importance. The selectivity filter (SF) of potassium channels functions as the C-type inactivation gate by alternating between conductive and non-conductive states. The bacterial KcsA potassium channel, characterized by well-defined structural features, serves as an ideal model for investigating this mechanism through molecular dynamics (MD) simulations. However, limitations in computational power and the time scales of C-type inactivation, which extend up to seconds, have constrained a comprehensive understanding of this process. This study used high-throughput steered molecular dynamics (SMD) simulations, employing a knowledge-based acceleration strategy, to capture spontaneous SF constriction within nanoseconds in KcsA. Over a thousand SMD simulations recorded hundreds of SF constriction events, revealing a common constriction mechanism driven by an ion occupancy switch from state 13 to state 14 within the SF, facilitated by water molecules located behind the SF. Simulations of the E71V-mutated KcsA suggest that this constricted state and mechanism may also extend to Kv-like channels, albeit with reduced water dependence. These findings underscore the essential roles of ions and water molecules in regulating protein dynamics and highlight strategies for high-throughput MD studies to further explore protein dynamics.

Infrared thermal imaging for assessing human perspiration and evaluating antiperspirant product efficacy

In humans, perspiration regulates core body temperature. Therefore, objectively evaluating it is essential for studying sweat gland function and mechanisms, particularly in antiperspirant efficacy studies. Various approaches have been developed for measuring human perspiration and evaluating antiperspirant efficacy, but are unsuitable for robust and routine clinical testing applications. This paper shows how infrared thermography, utilizing both high- and low-resolution modes, functions as a multiscale imaging modality. The high-resolution mode extracts physiological parameters (respiratory ~ 0.3 Hz and heart rate ~ 1.0 Hz) and visualizes the reduction of the sweat pore radii (from 359 ± 155 μm to 161 ± 47 μm) after antiperspirant application, consistent with known mechanisms of pore plugging and constriction induced by aluminum salts. The low-resolution mode quantitatively maps sweat retention in underarm clothing. All study participants in a clinical trial showed reduced sweat retention on their T-shirts due to antiperspirants, with reductions ranging from approximately 37–97% and an average reduction of 77.7 ± 22.1% using the developed methodology and tested antiperspirant. Overall, this non-invasive technique presents significant potential for clinical and personal care product evaluations, particularly in the early stages of product development.

The Degree of Capillary Absorption Reduction as a Parameter to Classify the Effectiveness of Secondary Horizontal Waterproofing

The dampness of the ground floors in buildings is generally a consequence of capillary rise of groundwater caused by the absence, damage or technical deterioration of the horizontal waterproofing of the masonry. Capillary absorption and damp transport are determined by the wetting properties of water relative to the material, as well as the structure and distribution of pores in the material. Chemical (injection) methods of secondary horizontal waterproofing are based on the technology of introducing injection liquid into the masonry section, which forms a lock which relies on the mechanism of capillary lumen constriction, hydrophilization, capillary sealing or a combined effect. A vital outcome of secondary horizontal waterproofing in a wall using the injection method is not only limited absorption but also its changed dynamics. The research paper describes the procedure leading to determining a criterion that allows to classify injection agents in terms of their impact on inhibiting capillary transport of damp in construction partitions.

Combining descriptive and predictive modeling to systematically design depth filtration-based harvest processes for biologics.

Advances in upstream production of biologics-particularly intensified fed-batch processes beyond 10% cell solids-have severely strained harvest operations, especially depth filtration. Bioreactors containing high amounts of cell debris (more than 40% particles <10 µm in diameter) are increasingly common and drive the need for more robust depth filtration processes, while accelerated timelines emphasize the need for predictive tools to accelerate development. Both needs are constrained by the current limited mechanistic understanding of the harvest filter-feedstream system. Historically, process development relied on screening scale-down depth filter devices and conditions to define throughput before fouling, indicated by increasing differential pressure and/or particle breakthrough (measured via turbidity). This approach is straightforward, but resource-intensive, and its results are inherently limited by the variability of the feedstream. Semi-empirical models have been developed from first principles to describe various mechanisms of filter fouling, that is, pore constriction, pore blocking, and/or surface deposit. Fitting these models to experimental data can assist in identifying the dominant fouling mechanism. Still, this approach sees limited application to guide process development, as it is descriptive, not predictive. To address this gap, we developed a hybrid modeling approach. Leveraging historical bench scale filtration process data, we built a partial least squares regression model to predict particle breakthrough from filter and feedstream attributes, and leveraged the model to demonstrate prediction of filter performance a priori. The fouling models are used to interpret and provide physical meaning to these computational models. This hybrid approach-combining the mechanistic insights of fouling models and the predictive capability of computational models-was used to establish a robust platform strategy for depth filtration of Chinese hamster ovary cell cultures. As new data continues to teach the computational models, in silico tools will become an essential part of harvest process development by enabling prospective experimental design, reducing total experimental load, and accelerating development under strict timelines.

ATPase activity of DFCP1 controls selective autophagy

Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that is activated by membrane binding and dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 has a minor effect on bulk autophagic flux, DFCP1 is required to maintain the autophagic flux of p62 under both fed and starved conditions, and this is dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localize to forming omegasomes, these omegasomes fail to constrict properly in a size-dependent manner. Consequently, the release of nascent autophagosomes from large omegasomes is markedly delayed. While knockout of DFCP1 does not affect bulk autophagy, it inhibits selective autophagy, including aggrephagy, mitophagy and micronucleophagy. We conclude that DFCP1 mediates ATPase-driven constriction of large omegasomes to release autophagosomes for selective autophagy.

Mechanism of constriction in a high frequency pulsed welding arc

The phenomenon of arc constriction at high frequency pulsing was investigated experimentally using the monochromatic imaging method (MIM) on arcs generated with a thermionic electrode. Square pulses with 50% duty cycle between 75 A and 125 A (average of 100 A) were explored at frequencies of 1 kHz, 3 kHz, and 5 kHz. Constant current arcs of 75 A, 100 A, and 125 A were also investigated as a reference. The observations indicate that the high-temperature region of the arc (above 14,000 K) increase in volume during the pulse, to sizes larger than those corresponding to the pulse current applied in steady state (125 A), similarly during the background current, the size of the hot regions is smaller than under the same value of constant current (75 A). The size increase during the pulse is much more prominent than the decrease during the background. This net increase in the average size of the most electrically conductive region takes current away from the less conductive periphery of the arc. The pulse frequencies from 1 kHz to 5 kHz have significant effect on thermal delay from Joule region to convection region. The mechanisms identified explain the arc constriction observed experimentally. This new understanding allows for open arcs that can rival more complex plasma torches in heat concentration and in the ability to control the heat source from a welding arc using purely electronic means, without resorting to gas mixing or mechanical adjustments.

Toxoplasma gondii's Basal Complex: The Other Apicomplexan Business End Is Multifunctional.

The Apicomplexa are famously named for their apical complex, a constellation of organelles at their apical end dedicated to invasion of their host cells. In contrast, at the other end of the cell, the basal complex (BC) has been overshadowed since it is much less prominent and specific functions were not immediately obvious. However, in the past decade a staggering array of functions have been associated with the BC and strides have been made in understanding its structure. Here, these collective insights are supplemented with new data to provide an overview of the understanding of the BC in Toxoplasma gondii. The emerging picture is that the BC is a dynamic and multifunctional complex, with a series of (putative) functions. The BC has multiple roles in cell division: it is the site where building blocks are added to the cytoskeleton scaffold; it exerts a two-step stretch and constriction mechanism as contractile ring; and it is key in organelle division. Furthermore, the BC has numerous putative roles in ‘import’, such as the recycling of mother cell remnants, the acquisition of host-derived vesicles, possibly the uptake of lipids derived from the extracellular medium, and the endocytosis of micronemal proteins. The latter process ties the BC to motility, whereas an additional role in motility is conferred by Myosin C. Furthermore, the BC acts on the assembly and/or function of the intravacuolar network, which may directly or indirectly contribute to the establishment of chronic tissue cysts. Here we provide experimental support for molecules acting in several of these processes and identify several new BC proteins critical to maintaining the cytoplasmic bridge between divided parasites. However, the dispensable nature of many BC components leaves many questions unanswered regarding its function. In conclusion, the BC in T. gondii is a dynamic and multifunctional structure at the posterior end of the parasite.

Tracing back variations in archaeal ESCRT-based cell division to protein domain architectures.

The Endosomal Sorting Complex Required for Transport (ESCRT) system is a multi-protein machinery that is involved in cell division of both Eukaryotes and Archaea. This spread across domains of life suggests that a precursor ESCRT machinery existed already at an evolutionary early stage of life, making it a promising candidate for the (re)construction of a minimal cell division machinery. There are, however, only few experimental data about ESCRT machineries in Archaea, due to high technical challenges in cultivation and microscopy. Here, we analyse the proteins of ESCRT machineries in archaea bioinformatically on a protein domain level, to enable mechanistical comparison without such challenging experiments. First, we infer that there are at least three different cell division mechanisms utilizing ESCRT proteins in archaea, probably similar in their constriction mechanisms but different in membrane tethering. Second, we show that ESCRT proteins in the archaeal super-phylum Asgard are highly similar to eukaryotic ESCRT proteins, strengthening the recently developed idea that all Eukaryotes descended from archaea. Third, we reconstruct a plausible evolutionary development of ESCRT machineries and suggest that a simple ESCRT-based constriction machinery existed in the last archaeal common ancestor. These findings not only give very interesting insights into the likely evolution of cell division in Archaea and Eukaryotes, but also offer new research avenues by suggesting hypothesis-driven experiments for both, cell biology and bottom-up synthetic biology.

Gravity-driven membrane filtration of primary wastewater effluent for edible plant cultivations: Membrane performance and health risk assessment

In this study, gravity-driven membrane (GDM) systems packed with Icelandic lava stones (0%, 26% and 53%) were employed to treat primary wastewater for reuse. The three GDM systems could achieve > 75% removal of organics. While, the lava stone biocarriers facilitated higher nitrogen removals (>51% vs. ~37% without biocarriers), which were related to the presence of manganese oxide (MnOx) in the lava stones. A combined mechanism of pore constriction and cake fouling was predominant during flux stabilization, regardless of lava stone packing ratios. Compared with non-cleaning or geothermal water-/persulfate-based cleaning, sodium hypochlorite-based cleaning (0.5% in geothermal water at 50 °C, 10 min per 3–4 days) was more effective in improving flux recovery (~46–79%) and water permeability (~23–79%). Furthermore, GDM permeate was used to irrigate two vegetables (tomato and basil), with tap water and commercial fertilizer as comparison baselines. Statistically insignificant (p > 0.05) results were observed for vegetable growth profiles and non-essential heavy metal contents in soils. Even though the GDM permeate-irrigated vegetables had a slightly higher uptake of non-essential metal cadmium (Cd) compared to those with tap water, the hazard quotient index was still less than 1, indicating negligible human health risk.

Cell division in the archaeon Haloferax volcanii relies on two FtsZ proteins with distinct functions in division ring assembly and constriction.

In bacteria, the tubulin homologue FtsZ assembles a cytokinetic ring, termed the Z ring, and plays a key role in the machinery that constricts to divide the cells. Many archaea encode two FtsZ proteins from distinct families, FtsZ1 and FtsZ2, with previously unclear functions. Here, we show that Haloferax volcanii cannot divide properly without either or both FtsZ proteins, but DNA replication continues and cells proliferate in alternative ways, such as blebbing and fragmentation, via remarkable envelope plasticity. FtsZ1 and FtsZ2 colocalize to form the dynamic division ring. However, FtsZ1 can assemble rings independent of FtsZ2, and stabilizes FtsZ2 in the ring, whereas FtsZ2 functions primarily in the constriction mechanism. FtsZ1 also influenced cell shape, suggesting it forms a hub-like platform at midcell for the assembly of shape-related systems too. Both FtsZ1 and FtsZ2 are widespread in archaea with a single S-layer envelope, but archaea with a pseudomurein wall and division septum only have FtsZ1. FtsZ1 is therefore likely to provide a fundamental recruitment role in diverse archaea, and FtsZ2 is required for constriction of a flexible S-layer envelope, where an internal constriction force might dominate the division mechanism, in contrast with the single-FtsZ bacteria and archaea that divide primarily by wall ingrowth.

Lung tissue inflammatory response and pneumonocyte apoptosis of Sprague-Dawley rats after a 30-day exposure in methyl mercaptan vapor

ABSTRACT The objective of the research was to reveal the potential toxicity effects of methyl mercaptan on rat lung tissue. A dynamic exposure device and Sprague-Dawley (SD) rats were adopted. The exposure concentration of methyl mercaptan was 0.5 ± 0.1 ppm. The exposure procedure was 6 h/day, continuing for 30 days. The routine blood levels, oxidative stress levels in serum, immune molecule and cytokine in the serum and lung tissue were tested. Morphology injury of lung tissue was detected by Hematoxylin and Eosin (HE) staining. Apoptosis rate of alveolar epithelial cells were determined by TdT-mediated dUTP Nick End Labeling (TUNEL) assay. Reduction of body weight gain was observed in the male group during the exposure time, while there was no significant reduction of body weight gain in the female group. Pathological findings of terminal bronchiole constriction, alveolar congestion, and erythrocyte exudation confirmed the lung to be the main target organ. An apparent pneumonocyte apoptotic effection was also observed. Oxidative stress with lipid peroxidation, which affect blood antioxidant enzyme levels and induce apoptosis of alveolar epithelial cells, are recognized as a potential mechanism leading to terminal bronchiole constriction, alveoli congestion, and exudates of erythrocyte. Implications: The odor pollutants greatly affect the health of operation workers in the waste treatment plant, and odor complaints are becoming a major problem. The aim of this work is to identify the lung tissue inflammatory response of SD rats with chronic exposure to methyl mercaptan vapor at close to the recommended workplace concentration. In this study, we used a dynamic exposure device and chronic exposure model of rats to evaluate the potential toxicity effects of methyl mercaptan. The results showed that methyl mercaptan may cause lung inflammatory response and extensive lung cell apoptosis. Oxidative damage, with lipid peroxidation and alterations in blood antioxidant enzyme levels, was observed following methyl mercaptan exposure. This is recognized as a potential mechanism for terminal bronchiolar constriction, alveolar congestion, and erythrocyte exudation.

Simulations of Proposed Mechanisms of FtsZ-Driven Cell Constriction.

To divide, bacteria must constrict their membranes against significant force from turgor pressure. A tubulin homolog, FtsZ, is thought to drive constriction, but how FtsZ filaments might generate constrictive force in the absence of motor proteins is not well understood. There are two predominant models in the field. In one, FtsZ filaments overlap to form complete rings around the circumference of the cell, and attractive forces cause filaments to slide past each other to maximize lateral contact. In the other, filaments exert force on the membrane by a GTP-hydrolysis-induced switch in conformation from straight to bent. Here, we developed software, ZCONSTRICT, for quantitative three-dimensional (3D) simulations of Gram-negative bacterial cell division to test these two models and identify critical conditions required for them to work. We find that the avidity of any kind of lateral interactions quickly halts the sliding of filaments, so a mechanism such as depolymerization or treadmilling is required to sustain constriction by filament sliding. For filament bending, we find that a mechanism such as the presence of a rigid linker is required to constrain bending to within the division plane and maintain the distance observed in vivo between the filaments and the membrane. Of these two models, only the filament bending model is consistent with our lab's recent observation of constriction associated with a single, short FtsZ filament.IMPORTANCE FtsZ is thought to generate constrictive force to divide the cell, possibly via one of two predominant models in the field. In one, FtsZ filaments overlap to form complete rings which constrict as filaments slide past each other to maximize lateral contact. In the other, filaments exert force on the membrane by switching conformation from straight to bent. Here, we developed software, ZCONSTRICT, for three-dimensional (3D) simulations to test these two models. We find that a mechanism such as depolymerization or treadmilling are required to sustain constriction by filament sliding. For filament bending, we find that a mechanism that constrains bending to within the division plane is required to maintain the distance observed in vivo between the filaments and the membrane.

The mechanism of serotonin-induced constriction in 2K1C hypertensive rat mesenteric arteries

The mechanism of serotonin-induced constriction in 2K1C hypertensive rat mesenteric arteries

Augmentation of weld penetration by flux assisted TIG welding and its distinct variants for oxygen free copper

A comparative study to investigate the influences of single component fluxes on the depth-to-width ratio (DWR) of oxygen free copper was carried out with novel variants of tungsten inert gas (TIG) welding namely Activated TIG (A-TIG), Flux Bounded TIG (FB-TIG) and Flux Zoned TIG (FB-TIG) processes. The experiments to identify the fluxes delivering the higher DWRs in A-TIG welding among thirteen distinct fluxes were followed by the trials with FB-TIG and FZ-TIG employing those identified DWR fluxes. The fluxes which outperformed with all the techniques were MoO3 & MgO. Reversed Marangoni and arc constriction mechanisms were perceived to be opt for such an increase in DWR. Metallurgical characterization of the weldment indicated distinct grain morphologies and certain defects as well in the weld zones. The FZ-TIG welding is postulated to surpass all the techniques in terms of bringing about the upmost weld penetration.

Peculiarities of glow discharge constriction in helium

The work experimentally demonstrates the fundamental difference between the constriction in helium and other inert gases (argon, neon). Despite the visual similarity, during the constriction of the positive column of a glow discharge in helium, only a compression of the emission of spectral lines is observed. In the constricted helium discharge there is no formation of a thin channel of current which is evident from the bremsstrahlung measurements. This phenomenon can be called the optical constriction. Unlike neon and argon, where the current cord is formed because of losses of charged particles in the volume due to the dissociative recombination, the discharge in helium is controlled by the ambipolar diffusion. Moreover, while in neon and argon constriction arises in times of hundreds of microseconds—units of milliseconds after the ionization balance establishment, constricted discharge in helium forms in times about 2 min after the establishment of the thermal balance between the discharge tube and the environment. It was shown that in helium the main mechanism of constriction is the inhomogeneous heating of the neutral gas which leads to the redistribution of the neutrals over the volume, in contrast to argon and neon, where thermal effects are almost insignificant and constriction is due to the peculiarities of electron kinetics. The absence of constriction of the emission of spectral lines during the cooling of the tube walls is demonstrated. The interpretation of the observed phenomena is given on the basis of a simple qualitative model. Experiments in helium were performed at reduced pressure pR = 200 torr cm and reduced currents up to 150 mA cm−1.

Investigation on arc plasma, droplet, and molten pool behaviours in compulsively constricted WAAM

In order to study the special constriction effect and physical process features during compulsively constricted WAAM (CC-WAAM), the dynamic behaviours of arc, droplets, and molten pool were visually investigated. Possible interactions inside the narrow space were discussed to explain the mechanism of the compulsive constriction on ejected plasma and droplets. Based on the captured images, the strong radiative emission indicates that the ejected plasma was at high temperature at least 6000 K. As the current increases higher-temperature plasma jets were expected, which would lead to a larger plasma volume in the absence of constriction. The relationship between current and droplet diameters was preliminarily established to enable prediction of the droplet size. An important feature of small-size droplets (as low as 0.89 mm) offered CC-WAAM a great potential to improve the precision of the additive manufacturing layers, although the minimum width of the deposited layer is much larger than the droplet diameter due to liquid spreading and accumulation. The droplets were found to have a slight impact on the molten pool behaviours, which produces stable molten pool shapes. Under the wide-range parameters, the deposited layers showed good appearances, which indicates the good adaptability of this novel technology.

Effect of Oxide Fluxes in Activated TIG Welding of Stainless Steel 316LN to Low Activation Ferritic/Martensitic Steel (LAFM) Dissimilar Combination

The aim of current work is to study the effect of different oxide fluxes in activated TIG welding process of dissimilar welding between LAFM and 316LN on weld dimensions, macro- and microexamination, and microhardness. Different oxide fluxes, namely TiO2, Fe2O3, CuO, Co3O4, and HgO, were used for the experiment, and its results were compared with convention TIG welding. Flux paste was applied over the area to be welded using a paint brush at the middle portion of the specimen. All welding experiments were carried out on a bead on a plate and under the same welding conditions and parameters. The experimental result reveals that full-length penetration was achieved with the use of Co3O4 and TiO2 fluxes. This enhancement in depth of penetration is attributed to the reverse Marangoni effect and arc constriction mechanism. In this study, insight was also revealed pertaining to the depth-enhancing mechanism present during ATIG welding of LAFM and SS316LN joints.

Actomyosin contraction during cellularization is regulated in part by Src64 control of Actin 5C protein levels.

Src64 is required for actomyosin contraction during cellularization of the Drosophila embryonic blastoderm. The mechanism of actomyosin ring constriction is poorly understood even though a number of cytoskeletal regulators have been implicated in the assembly, organization, and contraction of these microfilament rings. How these cytoskeletal processes are regulated during development is even less well understood. To investigate the role of Src64 as an upstream regulator of actomyosin contraction, we conducted a proteomics screen to identify proteins whose expression levels are controlled by src64. Global levels of actin are reduced in src64 mutant embryos. Furthermore, we show that reduction of the actin isoform Actin 5C causes defects in actomyosin contraction during cellularization similar to those caused by src64 mutation, indicating that a relatively high level of Actin 5C is required for normal actomyosin contraction and furrow canal structure. However, reduction of Actin 5C levels only slows down actomyosin ring constriction rather than preventing it, suggesting that src64 acts not only to modulate actin levels, but also to regulate the actomyosin cytoskeleton by other means.

Endothelin‐1 Potentiates Capsaicin‐induced Constriction of Human Adipose Arterioles

Mechanosensitive Ca2+‐permeable channels of the transient receptor potential vanilloid (TRPV) family, have emerged as important regulators of vascular tone. TRPV1, the first identified member of the TRPV subfamily, has been extensively studied in rodent and other animal vascular beds. On perivascular sensory nerves and endothelial cells (EC), TRPV1 activation is shown to cause vasodilation; whereas activation of smooth muscle (VSMC) TRPV1 causes vasoconstriction. Their role, however, in the human vasculature remains poorly understood. We hypothesized that TRPV1 channels are expressed in the human vasculature and influence vascular tone upon activation. The objective of the current study was to characterize the expression and vasomotor function of TRPV1 channels in human coronary and adipose arterioles (HCAs &amp; HAAs respectively). RT‐PCR analysis revealed the presence of TRPV1 transcripts in HCAs and HAAs isolated from multiple subjects. Consistent with mRNA expression, TRPV1 was detected in membrane fractions of HCAs and HAAs using immunoblotting. Upon examination of the vasomotor effect of capsaicin (TRPV1‐selective agonist) on HAAs, we found that bath application of capsaicin (10−9 – 10−6 M) induced dose‐dependent constriction of both endothelium‐intact (31.6 ± 9% at 10−6 M; n=6) and denuded HAAs (42.6 ± 10.6% at 10−6 M; n=5), suggesting a vasoconstrictive role for VSMC TRPV1 channels in the human microcirculation. This response was inhibited by the TRPV1‐selective inhibitor, SB366791 (0.2 ± 0.1% at 10−6 M; n=6 intact vessels), thus demonstrating the specificity of capsaicin for TRPV1. To investigate the mechanism of capsaicin‐induced constriction, we examined changes in global intracellular calcium concentration ([Ca2+]i) in native human VSMC. Capsaicin (10−7–10−6 M) induced dose‐dependent increases in [Ca2+]i in native VSMC, which were inhibited by SB366791 (1 μM), again supporting a vasoconstrictive role for VSMC TRPV1. To further explore the physiological significance of TRPV1 channels in vasomotor control, we investigated if TRPV1 channel function is modulated by the physiological vasoconstrictor, endothelin‐1 (ET‐1). HAAs were preconstricted with ET‐1 to 10–15% of their baseline diameter before bath application of increasing doses of capsaicin (10−9 – 10−6 M). Capsaicin‐induced constriction was potentiated by ET‐1 in both endothelium‐intact (50.1 ± 6.7% at 10−6M; n=9) and denuded (52 ± 5.2% at 10−6 M; n=5) arterioles. These results indicate that TRPV1 channels are expressed and functional in the human microvasculature and that their activity is modulated by endogenous vasoconstrictors such as ET‐1. Future studies are required to elucidate the mechanism of ET‐1 induced potentiation of TRPV1‐mediated constriction.Support or Funding InformationSupport for this research was provided by the National Heart, Lung and Blood Institute Grant RO1‐HL 096647 and American Heart Association Grant 18PRE34060242/Ankush Korishettar/2018.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.