Pore-forming Membrane Proteins Research Articles

Migraine and gasdermin D: a new perspective on the inflammatory basis of migraine.

Migraine is a common and disabling primary headache disorder and inflammation is a proposed factor in the complex ethiology of the disease. Gasdermin D (GSDMD) is a membrane pore-forming protein acting through the caspase system. End result is cell death caused by leakage of intracellular components to extracellular space which also results in inflammation. Stemming from this knowledge, the potential role of GSDMD in migraine was investigated in this prospective study. This prospective study was conducted between September 2022 to April 2023. 47 patients with migraine were designated as the patient group, whereas 47 healthy volunteers were designated as the control group. Serum GSDMD levels of both groups were compared, with an additional comparison between migraine patients during symptom-free and attack periods. Migraine related characteristics of the patients were also included in the study. Median GSDMD levels of the patient and control group did not reveal a significant difference. Nausea, vomiting and severity of headache were found to be correlated with GSDMD levels in migraine patients. Patients with nausea revealed a higher GSDMD level compared to patients without nausea during both symptom-free and attack periods (p = 0.021 and p = 0.01, respectively). Nausea was correlated to higher GSDMD levels in the patient population during symptom-free period (p = 0.030). The severity of pain was positively correlated with GSDMD levels during the attack period (p < 0.001). Gasdermin family and GSDMD in particular are promising prospects for therapy in a wide spectrum of disorders. Gasdermin proteins are candidates to be the focus for future studies both related to pathogenesis and drug therapy in migraine and varying inflammatory-driven clinical pictures.

An aptamer-triggered hybridization chain reaction strategy for ultra-sensitive biological nanopore detection of aflatoxin B1

Biological nanopore refers to a type of pore-forming membrane proteins, providing a unique platform for single molecule detection of DNA, RNA, peptide, etc. The direct detection of small molecules, especially of food chemical contaminants by biological nanopore, remains challenging due to their fast translocation dynamics, trace amount, and the interference from the complex food matrix. The effective recognition and signal conversion methods need to be developed. Herein, we employ an aptamer-triggered hybridization chain reaction (AtHCR) strategy for the detection of a mycotoxin, aflatoxin B1 (AFB1) by an α-hemolysin nanopore. In the absence of AFB1, the formed dsDNA polymers in HCR show a low frequency of long-time blockage signals. The presence of AFB1 can inhibit the hybridization chain reaction through the interaction between AFB1 and its aptamer, so the remaining hairpins can easily block the nanopore and induce frequent long-time blockage signals. The results show that the event frequency of long-blockage has a linear relationship with the AFB1 concentration in the range of 6.6 × 10−1–6.6 × 102 pmol/L. The recognition and amplification effect of AtHCR allows nanopore to detect AFB1 at a detection limit of 0.54 pmol/L. Furthermore, the developed AtHCR strategy can help biological nanopore to detect AFB1 in corn sample with satisfactory accuracy. The recognition and amplification effect achieved by coupling AtHCR to biological nanopore offers an enzyme-free single-molecule technology for the rapid detection of food chemical contaminants.

Preparation and characterization of monoclonal antibodies against porcine gasdermin D protein

Pyroptosis is a newly discovered type of pro-inflammatory programmed cell death that plays a vital role in various processes such as inflammations, immune responses, and pathogen infections. As one of the main executioners of pyroptosis, gasdermin D (GSDMD) is a membrane pore-forming protein that typically exists in a self-inhibitory state. Once activated, GSDMD will be cleaved into an N-terminal fragment with pore-forming activity, becoming the key indicator of pyroptosis activation, and a C-terminal fragment. Although commercial antibodies against human and murine GSDMD proteins are currently available, their reactivity with porcine GSDMD (pGSDMD) is poor, which limits research on the biological functions of pGSDMD and pyroptosis in pigs in vivo and in vitro. Here, five monoclonal antibodies (mAbs) were prepared by immunizing BALB/c mice with procaryotically expressed full-length pGSDMD, all of which did not cross react with human and murine GSDMD proteins. Epitope mapping demonstrated that 15H6 recognizes amino acids (aa) at positions 28–34 of pGSDMD (LQTSDRF), 19H3 recognizes 257–260aa (PPQF), 23H10 and 27A10 recognize 78–82aa (GPFYF), and 25E2 recognizes 429–435aa (PPTLLGS). The affinity constant and isotype of 15H6, 19H3, 23H10, 27A10, and 25E2 mAbs were determined to be 1.32 × 10−9, 3.66 × 10−9, 9.04 × 10−9, 1.83 × 10−9, and 8.00 × 10−8 mol/L and IgG1/κ, IgG2a/κ, IgG2a/κ, IgG1/κ, and IgG1/κ, respectively. Heavy- and light-chain variable regions sequencing showed that the heavy-chain complementarity-determining region (CDR) sequences of all five mAbs are completely different, while the light-chain CDR sequences of the four mAbs that recognize the N-terminus of pGSDMD are identical. Our prepared mAbs provide valuable materials for studying pGSDMD function and pyroptosis.Key points• A total of five mouse anti-pGSDMD mAbs were prepared, of which four recognize the N-terminus of pGSDMD and one recognize its C-terminus.• The main performance parameters of the five mAbs, including epitope, antibody titer, affinity constant, isotype, and heavy- and light-chain CDR, were characterized.• All five mAbs specifically recognize pGSDMD protein and do not cross react with human and murine GSDMD proteins.

Sensitive detection of viable Cronobacter sakazakii by bioluminescent reporter phage emitting stable signals with truncated holin

As outbreaks of foodborne illness caused by the opportunistic pathogen Cronobacter sakazakii (Cs) continue to occur, particularly in infants consuming powdered infant formula (PIF), the need for sensitive, rapid, and easy-to-use detection of Cs from food and food processing environments is increasing. Here, we developed bioluminescent reporter bacteriophages for viable Cs-specific, substrate-free, rapid detection by introducing luciferase and its corresponding substrate-providing enzyme complex into the virulent phage ΦC01. Although the reporter phage ΦC01_lux, constructed by replacing non-essential genes for phage infectivity with a luxCDABE reporter operon, produced bioluminescence upon Cs infection, the emitted signal was quickly decayed due to the superior bacteriolytic activity of ΦC01. By truncating the membrane pore-forming protein holin and thus limiting its function, the bacterial lysis was delayed and the resultant engineered reporter phage ΦC01_lux_Δhol could produce a more stable and reliable bioluminescent signal. Accordingly, ΦC01_lux_Δhol was able to detect at least an average of 2 CFU/ml of Cs artificially contaminated PIF and Sunsik and food contact surface models within a total of 7 h of assays, including 5 h of pre-enrichment for Cs amplification. The sensitive, easy-to-use, and specific detection of live Cs with the developed reporter phage could be applied as a novel complementary tool for monitoring Cs in food and food-related environments for food safety and public health.

Control of Osmotic-Engine-Driven Liposomes Using Biological Nanopores

Liposome-based molecular robots that molecular systems are integrated into a giant liposome have been proposed; they are expected to be applied in the fields of medicine, environmental science, food science, and energy science. However, the performance of these molecular robotic components, including intelligence, sensors, and actuators, still hinders their practical use. In particular, the actuators used in the molecular robots, such as molecular motors, do not provide sufficient performance to move the giant liposomes. Hence, we propose an osmotic-engine-driven liposome and demonstrate the migration of liposomes in a microfluidic channel by applying a salt concentration difference between the front and rear of the liposome. Although the migration mechanism is simple and has the potential to provide sufficient mobility performance, control techniques for the movement speed and on/off switching are not established. Herein, we describe a speed control method of osmotic-engine-driven liposomes using pore-forming membrane proteins. In this study, we evaluated the effect of reconstituted α-hemolysin (αHL) nanopores on the water permeability through lipid bilayers. Thereafter, we demonstrated the change in displacement speeds of liposomes with and without nanopores. We expect the speed control method using nanopores to be applied to the liposome-based molecular robots.

Sub-micro- and nano-sized polyethylene terephthalate deconstruction with engineered protein nanopores

The identification or design of biocatalysts to mitigate the accumulation of plastics, including sub-micro- and nano-sized polyethylene terephthalate (nPET), is becoming a global challenge. Here we computationally incorporated two hydrolytic active sites with geometries similar to that of Idionella sakaiensis PET hydrolase, to fragaceatoxin C (FraC), a membrane pore-forming protein. FraCm1/m2 could be assembled into octameric nanopores (7.0 nm high × 1.6–6.0 nm entry), which deconstructed (40 °C, pH 7.0) nPET from GoodFellow, commodities and plastic bottles. FraCm1 and FraCm2 degrade nPET by endo- and exo-type chain scission. While FraCm1 produces bis(2-hydroxyethyl) terephthalate as the main product, FraCm2 yields a high diversity of oligomers and terephthalic acid. Mechanistic and biochemical differences with benchmark PET hydrolases, along with pore and nPET dynamics, suggest that these pore-forming protein catalytic nanoreactors do not deconstruct macro-PET but are promising in nanotechnology for filtering, capturing and breaking down nPET, for example, in wastewater treatment plants.

AIM2-mediated Immunity to PlasmodiumInfection in Hepatocytes

Abstract Malaria, caused by Plasmodiumparasites is a severe disease affecting millions of people around the world. Plasmodiumundergoes obligatory development and replication in the hepatocytes, before initiating the life-threatening clinical disease of blood-stage malaria. Although the innate immune responses that control Plasmodiuminfection and development in the liver are known to limit clinical malaria and transmission, the mechanism of such responses remain relatively unknown. Here we demonstrate that the DNA of Plasmodiumis sensed by AIM2 (absent in melanoma 2) receptors in the infected hepatocytes, resulting in Caspase-1 activation. However, we observed that Caspase-1 undergoes an unconventional and incomplete proteolytic processing in such hepatocytes, resulting in the activation of the membrane pore-forming protein, Gasdermin D (GSDMD), without the concurrent activation of the inflammasome-associated pro-inflammatory cytokines such as interleukin (IL)-1 and IL-18. Nevertheless, the AIM2-mediated Caspase-1 activation pathway induced the elimination of Plasmodium-infected hepatocytes and a resultant control of malaria in the liver. Supported by grants from NIH (R01 AI168307)

AIM2 sensors mediate immunity to Plasmodium infection in hepatocytes

Malaria, caused by Plasmodium parasites is a severe disease affecting millions of people around the world. Plasmodium undergoes obligatory development and replication in the hepatocytes, before initiating the life-threatening blood-stage of malaria. Although the natural immune responses impeding Plasmodium infection and development in the liver are key to controlling clinical malaria and transmission, those remain relatively unknown. Here we demonstrate that the DNA of Plasmodium parasites is sensed by cytosolic AIM2 (absent in melanoma 2) receptors in the infected hepatocytes, resulting in Caspase-1 activation. Remarkably, Caspase-1 was observed to undergo unconventional proteolytic processing in hepatocytes, resulting in the activation of the membrane pore-forming protein, Gasdermin D, but not inflammasome-associated proinflammatory cytokines. Nevertheless, this resulted in the elimination of Plasmodium-infected hepatocytes and the control of malaria infection in the liver. Our study uncovers a pathway of natural immunity critical for the control of malaria in the liver.

Determination of Gasdermin Pores.

The gasdermin family represents a type of membrane pore-forming proteins. The gasdermin family is extensively characterized as the executioner of pyroptotic cell death in mammals; recent studies suggest that gasdermin-like pore-forming proteins are also present in bacteria and fungi. In humans, gasdermin D (GSDMD) is activated through inter-domain cleavage by caspase-1 in the canonical inflammasome pathway and cytosolic LPS-activated caspase-4 or caspase-5. The cleavage disrupts the autoinhibition of GSDMD and liberates the N-terminal gasdermin-N domain that binds to membrane lipids and forms pores of an inner diameter of ~18nm on the membrane, responsible for cell pyroptosis. Here, we describe the methods of determining the phospholipid-binding and pore-forming activity of gasdermins in a robust in vitro system. We also introduce a method of specifically detecting the caspase-cleaved form of GSDMD in pyroptotic cells.

The Pathogenesis of African Trypanosomiasis.

African trypanosomes are bloodstream protozoan parasites that infect mammals including humans, where they cause sleeping sickness. Long-lasting infection is required to favor parasite transmission between hosts. Therefore, trypanosomes have developed strategies to continuously escape innate and adaptive responses of the immune system, while also preventing premature death of the host. The pathology linked to infection mainly results from inflammation and includes anemia and brain dysfunction in addition to loss of specificity and memory of the antibody response. The serum of humans contains an efficient trypanolytic factor, the membrane pore-forming protein apolipoprotein L1 (APOL1). In the two human-infective trypanosomes, specific parasite resistance factors inhibit APOL1 activity. In turn, many African individuals express APOL1 variants that counteract these resistance factors, enabling them to avoid sleeping sickness. However, these variants are associated with chronic kidney disease, particularly in the context of virus-induced inflammation such as coronavirus disease 2019. Vaccination perspectives are discussed.

Lipid bilayer on a microdroplet integrated with a patterned Ag/AgCl microelectrode for voltage-clamp fluorometry of membrane transport

Voltage-clamp fluorometry (VCF) has been combined with artificial lipid bilayer systems to optically measure the dynamics and contribution of membrane proteins on molecular transport at the clamped membrane potential. However, the previous methods for VCF require expensive apparatus, such as total internal reflection fluorescence microscope, or require extensive experience for voltage-clamping, or possibly lead to imprecise membrane potential. This study describes VCF performed using a pico-liter-sized lipid bilayer chamber with an embedded Ag/AgCl microelectrode. Owing to the size of the picoliter order, molecular transport through the lipid bilayer can be detected using a normal fluorescent microscope. Easy electrical access into the lipid bilayer chamber can be realized because the microelectrode is spontaneously embedded inside when forming the lipid bilayer chamber. The microelectrode is made of Ag/AgCl; therefore, it does not tend to polarize, which results in reliable regulation of the membrane potential. Using the developed system, we performed the VCF of molecular transport through the lipid bilayer by incorporating the pore-forming membrane protein, α-hemolysin. This proposed method will increase the number of researches that can perform the VCF of molecular transport through artificial lipid bilayers.

Posttranslational and Therapeutic Control of Gasdermin-Mediated Pyroptosis and Inflammation.

Pyroptosis is a proinflammatory form of cell death, mediated by membrane pore-forming proteins called gasdermins. Gasdermin pores allow the release of the pro-inflammatory cytokines IL-1β and IL-18 and cause cell swelling and cell lysis leading to release of other intracellular proteins that act as alarmins to perpetuate inflammation. The best characterized, gasdermin D, forms pores via its N-terminal domain, generated after the cleavage of full length gasdermin D by caspase-1 or -11 (caspase-4/5 in humans) typically upon sensing of intracellular pathogens. Thus, gasdermins were originally thought to largely contribute to pathogen-induced inflammation. We now know that gasdermin family members can also be cleaved by other proteases, such as caspase-3, caspase-8 and granzymes, and that they contribute to sterile inflammation as well as inflammation in autoinflammatory diseases or during cancer immunotherapy. Here we briefly review how and when gasdermin pores are formed, and then focus on emerging endogenous mechanisms and therapeutic approaches that could be used to control pore formation, pyroptosis and downstream inflammation.

Non-extensitivity and criticality of atomic hydropathicity around a voltage-gated sodium channel’s pore: a modeling study

Voltage-gated sodium channels (NavChs) are pore-forming membrane proteins that regulate the transport of sodium ions through the cell membrane. Understanding the structure and function of NavChs is of major biophysical, as well as clinical, importance given their key role in cellular pathophysiology. In this work, we provide a computational framework for modeling system-size-dependent, i.e., cumulative, atomic properties around a NavCh’s pore. We illustrate our methodologies on the bacterial NavAb channel captured in a closed-pore state where we demonstrate that the atomic environment around its pore exhibits a bi-phasic spatial organization dictated by the structural separation of the pore domains (PDs) from the voltage-sensing domains (VSDs). Accordingly, a mathematical model describing packing of atoms around NavAb’s pore is constructed that allows—under certain conservation conditions—for a power-law approximation of the cumulative hydropathic dipole field effect acting along NavAb’s pore. This verified the non-extensitivity hypothesis for the closed-pore NavAb channel and revealed a long-range hydropathic interactions law regulating atom-packing around the NavAb’s selectivity filter. Our model predicts a PDs-VSDs coupling energy of sim !282.1 kcal/mol corresponding to a global maximum of the atom-packing energy profile. Crucially, we demonstrate for the first time how critical phenomena can emerge in a single-channel structure as a consequence of the non-extensive character of its atomic porous environment.

The Kv1.3 ion channel acts as a host factor restricting viral entry.

Virus entry into cells is the initial stage of infection and involves multiple steps, and interfering viral entry represents potential antiviral approaches. Ion channels are pore-forming membrane proteins controlling cellular ion homeostasis and regulating many physiological processes, but their roles during viral infection have rarely been explored. Here, the functional Kv1.3 ion channel was found to be expressed in human hepatic cells and tissues. The Kv1.3 was then revealed to restrict HCV entry via inhibiting endosome acidification-mediated viral membrane fusion. The Kv1.3 was also demonstrated to inhibit DENV and ZIKV with an endosome acidification-dependent entry, but have no effect on SeV with a neutral pH penetration. A Kv1.3 antagonist PAP-1 treatment accelerated animal death in ZIKV-infected Ifnar1-/- mice. Moreover, Kv1.3-deletion was found to promote weight loss and reduce survival rate in ZIKV-infected Kv1.3-/- mice. Altogether, the Kv1.3 ion channel behaves as a host factor restricting viral entry. These findings broaden understanding about ion channel biology.

Thrombin receptor PAR4 drives canonical NLRP3 inflammasome signaling in the heart.

The deleterious effects of diabetes in the heart are increasingly attributed to inflammatory signaling through the NLRP3 (NOD, LRR and PYD domains-containing protein 3) inflammasome. Thrombin antagonists reduce cardiac remodeling and dysfunction in diabetic mice, in part by suppressing fibrin-driven inflammation. The role of cellular thrombin receptor subtypes in this context is not known. We sought to determine the causal involvement of protease-activated receptors (PAR) in inflammatory signaling of the diabetic heart. Mice with diet-induced diabetes showed increased abundance of pro-caspase-1 and pro-interleukin (IL)-1β in the left ventricle (LV), indicating transcriptional NLRP3 inflammasome priming, and augmented cleavage of active caspase-1 and IL-1β, pointing to canonical NLRP3 inflammasome activation. Caspase-11 activation, which mediates non-canonical NLRP3 inflammasome signaling, was not augmented. Formation of the plasma membrane pore-forming protein N-terminal gasdermin D (GDSMD), a prerequisite for IL-1β secretion, was also higher in diabetic vs. control mouse LV. NLRP3, ASC and IL-18 expression did not differ between the groups, nor did expression of PAR1 or PAR2. PAR3 was nearly undetectable. LV abundance of PAR4 by contrast increased with diabetes and correlated positively with active caspase-1. Genetic deletion of PAR4 in mice prevented the diet-induced cleavage of caspase-1, IL-1β and GDSMD. Right atrial appendages from patients with type 2 diabetes also showed higher levels of PAR4, but not of PAR1 or PAR2, than non-diabetic atrial tissue, along with increased abundance of cleaved caspase-1, IL-1β and GSDMD. Human cardiac fibroblasts maintained in high glucose conditions to mimic diabetes also upregulated PAR4 mRNA and protein, and increased PAR4-dependent IL-1β transcription and secretion in response to thrombin, while PAR1 and PAR2 expressions were unaltered. In conclusion, PAR4 drives caspase-1-dependent IL-1β production through the canonical NLRP3 inflammasome pathway in the diabetic heart, providing mechanistic insights into diabetes-associated cardiac thromboinflammation. The emerging PAR4-selective antagonists may provide a feasible approach to prevent cardiac inflammation in patients with diabetes.

Acid-sensing ion channels blockade attenuates pressor and sympathetic responses to skeletal muscle metaboreflex activation in humans.

In animals, the blockade of acid-sensing ion channels (ASICs), cation pore-forming membrane proteins located in the free nerve endings of group IV afferent fibers, attenuates increases in arterial pressure (AP) and sympathetic nerve activity (SNA) during muscle contraction. Therefore, ASICs play a role in mediating the metabolic component (skeletal muscle metaboreflex) of the exercise pressor reflex in animal models. Here we tested the hypothesis that ASICs also play a role in evoking the skeletal muscle metaboreflex in humans, quantifying beat-by-beat mean AP (MAP; finger photoplethysmography) and muscle SNA (MSNA; microneurography) in 11 men at rest and during static handgrip exercise (SHG; 35% of the maximal voluntary contraction) and postexercise muscle ischemia (PEMI) before (B) and after (A) local venous infusion of either saline or amiloride (AM), an ASIC antagonist, via the Bier block technique. MAP (BAM +30 ± 6 vs. AAM +25 ± 7 mmHg, P = 0.001) and MSNA (BAM +14 ± 9 vs. AAM +10 ± 6 bursts/min, P = 0.004) responses to SHG were attenuated under ASIC blockade. Amiloride also attenuated the PEMI-induced increases in MAP (BAM +25 ± 6 vs. AAM +16 ± 6 mmHg, P = 0.0001) and MSNA (BAM +16 ± 9 vs. AAM +8 ± 8 bursts/min, P = 0.0001). MAP and MSNA responses to SHG and PEMI were similar before and after saline infusion. We conclude that ASICs play a role in evoking pressor and sympathetic responses to SHG and the isolated activation of the skeletal muscle metaboreflex in humans. NEW & NOTEWORTHY We showed that regional blockade of the acid-sensing ion channels (ASICs), induced by venous infusion of the antagonist amiloride via the Bier block anesthetic technique, attenuated increases in arterial pressure and muscle sympathetic nerve activity during both static handgrip exercise and postexercise muscle ischemia. These findings indicate that ASICs contribute to both pressor and sympathetic responses to the activation of the skeletal muscle metaboreflex in humans.

Abstract 1309: Targeting GluN2C ion channels as a promising approach in the treatment of melanoma

Abstract Ion channels are pore-forming integral membrane proteins that are known to have an impact on all hallmarks of cancer. Due to their expression on the surface of the cells, ion channels provide a distinct therapeutic target with more facilitated access from the extracellular milieu. NMDA receptors are a subtype of ligand-gated ion channels that express unique features including high calcium permeability as well as the requirement for binding of glutamate and the co-agonist glycine in order to be activated. Here we show that a glutamate ionotropic NMDA (N-methyl-D-aspartate) type ion channel, GluN2C is critically required for survival of human melanoma cells. Publicly available microarray data from Gene Expression Omnibus (GEO) were acquired in order to identify the differentially expressed ion channel genes in 127 melanoma patient samples relative to benign nevi or human epidermal melanocytes. Data exists into two major cohorts: First cohort consisted of 18 benign nevi and 45 melanoma (GSE3189), while second cohort contained 8 melanocytes and 82 melanoma (GSE29377). A bioinformatics approach was undertaken to determine commonly upregulated ion channels between the two cohorts. Subsequently, functional enrichment analysis along with pathway evaluation of these genes revealed GluN2C as a novel candidate for further investigation in this study. Upregulation of GluN2C was confirmed in a panel of melanoma cell lines and fresh melanoma isolates. Strikingly, silencing of GluN2C markedly reduced viability and clonogenicity in melanoma cells. Consistently, a pharmacological inhibitor of GluN2C potently killed melanoma cells with minimal effects on melanocytes and fibroblasts. Killing of melanoma cells by inhibition of GluN2C was not due to induction of apoptosis, since a general caspase inhibitor Z-VAD-FMK failed to rescue cells from dying. In contrast, the late stage autophagy inhibitor Bafilomycin A1 blocked the GluN2C induced cell death suggesting that killing of melanoma cells following inhibition of GluN2C is in part associated with induction of autophagy. Collectively, these results uncover a potential role for GluN2C ion channels in the pathogenesis of melanoma and suggest that these ion channels may constitute a promising target for melanoma treatment. Citation Format: Hessam Tabatabaee, Yuchen Feng, Hamed Yari, Ting La, Simonne Sherwin, Lei Jin, Rebecca Lim, Alan Brichta, Xu Dong Zhang. Targeting GluN2C ion channels as a promising approach in the treatment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1309.

The dynamics of K+ channel gates as a biological transistor

Potassium channels are pore-forming membrane proteins that open and close in response to changes in a chemical or electrical potential, thereby regulating the flow of potassium ions across biological membranes. Two regions of the same channels are acting in tandem and enable ion flow through the channel pore. I refer to this coupled action as a “gate linker”. To closely examine the role of the gate linker in the channel function, I mutated the amino acids in the cDNA of this region, and used from knowen mutaion, either alone or together with the amino acids of adjacent regions.I have emphasized the importance of the linker between these two gates - mutations in this region may cause conformational changes that play a fundamental role in mediating the coupling between the voltage sensor, activation gate and selectivity filter elements of Kv channels. I observe that free energy considerations show the significance of the coupling between the activation and inactivation gates. Moreover, a symmetry between the coupling and sensor spring strength leads to the destruction of ion conductivity. I present a thermodynamic framework for the possible study of multiple channel blocks. The arising physical perspective of the gating process gives rise to new research avenues of the coupling mode of potassium channels and may assist in explaining the centrality of the “gate linker” to the channel function.

One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells.

Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant unilamellar vesicles (GUVs), produced at the milliliter scale directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Toward this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil–surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein α-hemolysin and assess the membrane quality with cryotransmission electron microscopy (cryoTEM), fluorescence recovery after photobleaching (FRAP), and zeta-potential measurements as well as confocal fluorescence imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques, offering high volumes, a flexible choice of lipid compositions and buffer conditions, straightforward coreconstitution of proteins, and a high encapsulation efficiency of biomolecules and even large cargo including cells. We thereby provide a simple, robust, and broadly applicable strategy to mass-produce complex multicomponent GUVs for high-throughput testing in synthetic biology and biomedicine, which can directly be implemented in laboratories around the world.

Acid‐Sensing Ion Channels Blockade Attenuates Pressor and Sympathetic Responses to Skeletal Muscle Metaboreflex Activation in Humans

In animals, the blockade of acid‐sensing ion channels (ASICS), cation pore‐forming membrane proteins located in the free nerve endings of group IV afferent fibers that are activated by acidic pH transients, attenuates reflex increases in sympathetic nerve activity (MSNA) and blood pressure (BP) during muscle contraction. These findings indicate that ASICS have an important role in mediating the metabolic component (skeletal muscle metaboreflex) of the exercise pressor reflex in animal models. Nonetheless, it is still unclear whether these findings can be translated to humans. We then tested the hypothesis that ASICS play a role in evoking the skeletal muscle metaboreflex in humans, quantifying beat‐by‐beat mean BP (MBP, Finometer), MSNA (peroneal nerve, microneurography) and heart rate (HR, ECG) in 11 healthy young men (N = 11) during rest, static handgrip exercise (SHE, 2 min at 35% of the MVC) and post‐exercise muscle ischemia (PEMI, 2 min) before (BFA) and after local infusion of amiloride (AFA −0.75 mg/mL), an antagonist of ASICs, in saline (2.5% of total forearm volume) via modified Bier block (regional intravenous anesthesia). In an additional protocol, participants (N = 7) underwent the experimental procedures before (BFS) and after (AFS) venous infusion of a similar amount of saline via modified Bier block. MBP responses were slightly but significantly attenuated during SHE after amiloride infusion (BFA +29.6 ± 6.1 vs. AFA +24.3 ± 7.0 mmHg, p = 0.001). A smaller increase in MSNA (N = 9) was also observed as a consequence of ASICs blockade (BFA +14.5 ± 9.6 vs. AFA +10.7 ± 6.5 bursts/min, p = 0.004). Equivalent SHE‐induced increases in HR were observed before and after ASICs blockade (p &gt; 0.05). Amiloride infusion attenuated the PEMI‐induced increases in both MBP (BFA +25.8 ± 6.9 vs. AFA +16.3 ± 6.4 mmHg, p = 0.0001) and MSNA (BFA +16.2 ± 9.2 vs. AFA +8.4 ± 8.0 bursts/min, p = 0.0001). In the additional protocol, SHE‐evoked increases in MBP (BFS +28.7 ± 12.2 vs. AFS +27.0 ± 9.8 mmHg, p = 0.477), MSNA (N = 5, BFS +10.0 ± 8.6 vs. AFS +13.0 ± 9.4 burst/min, p = 0.208) and HR (BFS +32.0 ± 12.4 vs. AFS +37.2 ± 22.5 bpm/min, p = 0.265) did not differ before and after saline infusion. Similar observations were obtained for MBP (BFS +24.2 ± 14.2 vs. AFS +23.7 ± 10.4 mmHg, p = 0.830) and MSNA (BFS +10.0 ± 8.6 vs. AFS +13.0 ± 9.4 burst/min, p = 0.208) during PEMI. Therefore, these findings indicate that ASICs play a role in evoking pressor and sympathetic responses to both SHE and isolated activation of the skeletal muscle metaboreflex in humans.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.