Endogenous Channels Research Articles

A synthetic method to assay polycystin channel biophysics.

Ion channels are biological transistors that control ionic flux across cell membranes to regulate electrical transmission and signal transduction. They are found in all biological membranes and their conductive state kinetics are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident ionophores. Here, we describe a fully synthetic method to assay functional properties of polycystin channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their oligomeric assembly, membrane integration, orientation, and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate channels resistant to biophysical analysis and pharmacological characterization.

A synthetic method to assay polycystin channel biophysics

Ion channels are biological transistors that control ionic flux across cell membranes to regulate electrical transmission and signal transduction. They are found in all biological membranes and their conductive state kinetics are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident ionophores. Here, we describe a fully synthetic method to assay functional properties of polycystin channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their oligomeric assembly, membrane integration, orientation, and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate channels resistant to biophysical analysis and pharmacological characterization.

P2X7R-primed keratinocytes are susceptible to apoptosis via GPCR-Gβγ-pERK signal pathways

BackgroundCell death constitutes a pivotal biological phenomenon essential for the preservation of homeostasis within living organisms. In the context of maintaining a functional skin barrier, keratinocytes exert positively and negatively control cell death signals. However, in patients with severe drug eruptions, anomalous overexpression of the formyl peptide receptor 1 (FPR1) in keratinocytes elicits a distinctive mode of cell death known as necroptosis, thereby suffering a loss of the skin barrier. The precise molecular mechanisms connecting FPR1 activation to this cell death remain unclear. ObjectiveWe have investigated the intracellular signal transduction cascade governing FPR1-mediated cell death in cultured keratinocytes. MethodsWe used HaCaT cells as a model keratinocyte. The expression of FPR1 was detected with qPCR. The presence of cell death events was monitored through live-cell fluorescent staining and LDH release assays. Furthermore, the phosphorylation of ERK was assessed via Western blot analysis. Intracellular signal pathways were investigated using specific inhibitors. ResultsLigand stimulation of an endogenous ion channel, purinergic receptor P2X7 (P2X7R), increased the FPR1 expression level. This upregulated FPR1 demonstrated functional competence in the phosphorylation of downstream MAP kinase and the initiation of cell death. Notably, this cell death was ameliorated upon the administration of inhibitors targeting Gβγ, ERK, and caspases. ConclusionThe induction and stimulation of FPR1 initiated apoptosis in keratinocytes via the Gβγ-pERK signaling pathway. Our findings postulate that the downstream components of FPR1 represent an alternative therapeutic target for preventing unintended keratinocyte cell death.

Trust in Lending

Abstract We develop a theory of trust in lending that distinguishes between reputation and trust. Banks emerge as more trusted lenders than non-banks. We show that trust severs the link between performance and the cost and availability of financing for lenders, but trust can be lost and is difficult to regain. Banks survive an erosion of trust better than non-banks. Banks' trust advantage arises from the lower cost of funding due to insured deposits and an endogenous belief revision channel that complements the effect of the funding cost advantage. The results have novel policy relevance for deposit insurance scope.

Long-term changes in transmembrane voltage after electroporation are governed by the interplay between nonselective leak current and ion channel activation

Electroporation causes a temporal increase in cell membrane permeability and leads to prolonged changes in transmembrane voltage (TMV) in both excitable and non-excitable cells. However, the mechanisms of these TMV changes remain to be fully elucidated. To this end, we monitored TMV over 30 min after exposing two different cell lines to a single 100 µs electroporation pulse using the FLIPR Membrane Potential dye. In CHO-K1 cells, which express very low levels of endogenous ion channels, membrane depolarization following pulse exposure could be explained by nonselective leak current, which persists until the membrane reseals, enabling the cells to recover their resting TMV. In U-87 MG cells, which express many different ion channels, we unexpectedly observed membrane hyperpolarization following the initial depolarization phase, but only at 33 °C and not at 25 °C. We developed a theoretical model, supported by experiments with ion channel inhibitors, which indicated that hyperpolarization could largely be attributed to the activation of calcium-activated potassium channels. Ion channel activation, coupled with changes in TMV and intracellular calcium, participates in various physiological processes, including cell proliferation, differentiation, migration, and apoptosis. Therefore, our study suggests that ion channels could present a potential target for influencing the biological response after electroporation.

Using Localization Microscopy to Quantify Calcium Channels at Presynaptic Boutons.

Calcium channels at synaptic boutons are critical for synaptic function, but their number and distribution are poorly understood. This gap in knowledge is primarily due to the resolution limits of fluorescence microscopy. In the last decade, the diffraction limit of light was surpassed, and fluorescent molecules can now be localized with nanometer precision. Concurrently, new gene editing strategies allowed direct tagging of the endogenous calcium channel genes-expressed in the correct cells and at physiological levels. Further, the repurposing of self-labeling enzymes to attach fluorescent dyes to proteins improved photon yields enabling efficient localization of single molecules. Here, we describe tagging strategies, localization microscopy, and data analysis for calcium channel localization. In this case, we are imaging calcium channels fused with SNAP or HALO tags in live anesthetized C. elegans nematodes, but the analysis is relevant for any super-resolution preparations. We describe how to process images into localizations and protein clusters into confined nanodomains. Finally, we discuss strategies for estimating the number of calcium channels present at synaptic boutons. Key features • Super-resolution imaging of live anesthetized C. elegans. • Three-color super-resolution reconstruction of synapses. • Nanodomains and the distribution of proteins. • Quantification of the number of proteins at synapses from single-molecule localization data.

A synthetic method to assay polycystin channel biophysics.

Ion channels are biological transistors that control ionic flux across cell membranes to regulate electrical transmission and signal transduction. They are found in all biological membranes and their conductive state kinetics are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident ionophores. Here we describe a fully synthetic method to assay functional properties of polycystin channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their oligomeric assembly, membrane integration, orientation and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate channels resistant to biophysical analysis and pharmacological characterization.

Remote neurostimulation through an endogenous ion channel using a near-infrared light-activatable nanoagonist.

The development of noninvasive approaches to precisely control neural activity in mammals is highly desirable. Here, we used the ion channel transient receptor potential ankyrin-repeat 1 (TRPA1) as a proof of principle, demonstrating remote near-infrared (NIR) activation of endogenous neuronal channels in mice through an engineered nanoagonist. This achievement enables specific neurostimulation in nongenetically modified mice. Initially, target-based screening identified flavins as photopharmacological agonists, allowing for the photoactivation of TRPA1 in sensory neurons upon ultraviolet A/blue light illumination. Subsequently, upconversion nanoparticles (UCNPs) were customized with an emission spectrum aligned to flavin absorption and conjugated with flavin adenine dinucleotide, creating a nanoagonist capable of NIR activation of TRPA1. Following the intrathecal injection of the nanoagonist, noninvasive NIR stimulation allows precise bidirectional control of nociception in mice through remote activation of spinal TRPA1. This study demonstrates a noninvasive NIR neurostimulation method with the potential for adaptation to various endogenous ion channels and neural processes by combining photochemical toolboxes with customized UCNPs.

The Effect of Calcium Ions on the Electrophysiological Properties of Single ANO6 Channels.

Proteins belonging to the anoctamin (ANO) family form calcium-activated chloride channels (CaCCs). The most unusual member of this family, ANO6 (TMEM16F), simultaneously exhibits the functions of calcium-dependent scramblase and the ion channel. ANO6 affects the plasma membrane dynamics and phosphatidylserine transport; it is also involved in programmed cell death. The properties of ANO6 channels remain the subject of debate. In this study, we investigated the effect of variations in the intracellular and extracellular concentrations of calcium ions on the electrophysiological properties of endogenous ANO6 channels by recording single ANO6 channels. It has been demonstrated that (1) a high calcium concentration in an extracellular solution increases the activity of endogenous ANO6 channels, (2) the permeability of endogenous ANO6 channels for chloride ions is independent of the extracellular concentration of calcium ions, (3) that an increase in the intracellular calcium concentration leads to the activation of endogenous ANO6 channels with double amplitude, and (4) that the kinetics of the channel depend on the plasma membrane potential rather than the intracellular concentration of calcium ions. Our findings give grounds for proposing new mechanisms for the regulation of the ANO6 channel activity by calcium ions both at the inner and outer sides of the membrane.

A liquidity preference approach to nonfinancial corporate liquid asset holdings

This paper examines the long-run pattern of the U.S. nonfinancial corporate sector’s liquid financial asset holdings in the period from 1951 to 2018. My approach to examining this pattern builds from the banks’ liquidity preference theory put forward in Keynes’ 1937 papers, as well as Minsky’s 1957 paper discussing the role of financial innovation in the banks’ management of liquidity preference. I argue that financial intermediaries affect nonfinancial corporations’ liquid asset holdings through two primary money creation channels: (1) banks’ commercial and industrial loans advanced to nonfinancial corporations and (2) the liquidity premium of financial intermediaries, which can be measured by the interest rate spread in the intermediaries’ liabilities markets. Through theoretical and empirical exercises, I find that these two endogenous liquidity creation channels have been the important factors accounting for liquid asset accumulation by the nonfinancial corporate sector. Overall, my results suggest that financial innovations, structural changes of the U.S. financial system, and financial intermediaries’ liquidity premium could contribute to our understanding of the shift in nonfinancial corporations’ asset portfolio composition in favor of liquid financial assets.

Tracking endogenous proteins based on RNA editing-mediated genetic code expansion.

Protein labeling approaches are important to study proteins in living cells, and genome editing tools make it possible to tag endogenous proteins to address the concerns associated with overexpression. Here we established RNA editing-mediated noncanonical amino acids (ncAAs) protein tagging (RENAPT) to site-specifically label endogenous proteins with ncAAs in living cells. RENAPT labels protein in a temporary and nonheritable manner and is not restricted by protospacer adjacent motif sequence. Using a fluorescent ncAA or ncAA with a bio-orthogonal reaction handle for subsequent dye labeling, we demonstrated that a variety of endogenous proteins can be imaged at their specific subcellular locations. In addition, two proteins can be tagged individually and simultaneously using two different ncAAs. Furthermore, endogenous ion channels and neuron-specific proteins can be real-time labeled in primary neurons. Thus, RENAPT presents a promising platform with broad applicability for tagging endogenous proteins in living cells to study their localization and functions.

Extracellular membrane vesicles derived from Komagataeibacter oboediens exposed on the International Space Station fuse with artificial eukaryotic membranes in contrast to vesicles of reference bacterium

Membranous Extracellular Vesicles (EVs) of Gram-negative bacteria are a secretion and delivery system that can disseminate bacterial products and interact with hosts and the environment. EVs of nonpathogenic bacteria deliver their contents by endocytosis into eukaryotic cells, however, no evidence exists for a fusion delivery mechanism. Here, we describe the fusion of exposed to space/Mars-like stressors simulated on the International Space Station vesicles (E-EVs) from Komagataeibacter oboediens to different types of model planar membranes in comparison with the EVs of the ground-based reference strain. The most reliable fusion was achieved with PC:PE:ergosterol or sterol-free PC:PE bilayers. The relative permeability ratio (PK+/PCl-) estimated from the shift of zero current potential according to Goldman–Hodgkin–Katz equation consisted of 4.17 ± 0.48, which coincides with preferential cation selectivity of the EV endogenous channels. The increase in membrane potential from 50 mV to 100 mV induced the fusion of E-EVs with all tested lipid compositions. The fusion of model exosomes with planar bilayer lipid membranes was confirmed by separate step-like increases in its conductance. In contrast, the ground-based reference K. oboediens EVs never induced the fusion event. In our study, we show membrane lipidome perturbations and increased protein aggregation occurred in the exposed samples in the harsh environment when outer membranes of K. oboediens acquired the capability of both homo- and heterotypic fusion possibly by altered membrane fluidity and the pore-forming capability.

Calcium chelation independent effects of BAPTA on endogenous ANO6 channels in HEK293T cells

Selective calcium chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) is a common tool to investigate calcium signaling. However, BAPTA express various effects on intracellular calcium signaling, which are not related to its ability to bind Ca2+. In patch clamp experiments, we investigated calcium chelation independent effects of BAPTA on endogenous calcium-activated chloride channels ANO6 (TMEM16F) in HEK293T cells. We have found that application of BAPTA to intracellular solution led to two distinct effects on channels properties. On the one hand, application of BAPTA acutely reduced amplitude of endogenous ANO6 channels induced by 10 μM Ca2+ in single channel recordings. On the other hand, BAPTA application by itself induced ANO6 channel activity in the absence of the intracellular calcium elevation. Open channel probability was enhanced by increasing the intracellular BAPTA concentration from 0.1 to 1 and 10 mM. Another calcium chelator EGTA did not demonstrate chelation independent effects on the ANO6 activity in the same conditions. Due to off target effects BAPTA should be used with caution when studying calcium-activated ANO6 channels.

Enhanced Methodologies forInvestigating the Electrophysiological Characteristics of Endogenous Pannexin 1 Intercellular Cell-Cell Channels.

Gap junctions, pivotal intercellular conduits, serve as communication channels betweenadjacent cells, playing a critical role in modulating membrane potential distribution across cellular networks. The family ofPannexin (Panx) proteins, in particular Pannexin1 (Panx1), are widelyexpressed in vertebrate cells and exhibit sequence homology with innexins, the invertebrate gap junction channelconstituents. Despite being ubiquitously expressed, detailed functional and pharmacological properties ofPanx1 intercellular cell-cell channelsrequire further investigation. In this chapter, we introduce optimized cell culture methodologies and electrophysiology protocols to expedite the exploration of endogenous Panx1 cell-cell channels in TC620 cells, a human oligodendroglioma cell line that naturally expresses Panx1. We anticipate theserefined protocols will significantly contributeto future characterizations of Panx1-based intercellularcell-cell channelsacross diverse cell types andoffer valuable insights into both normalcellular physiology and pathophysiology.

Expression and Functional Analysis of Ctenophore Glutamate Receptor Genes.

The functional analysis of ctenophore neurotransmitter receptors, transporters, and ion channels can be greatly simplified by use of heterologous expression systems. Heterologous expression allows the characterization of individual membrane proteins, expressed at high levels in cells, where background activity by endogenous ion channels and transporters is with few exceptions minimal. The goal of such experiments is to gain an in-depth understanding of the behavior and regulation of individual molecular species, which is challenging in native tissue, but especially so in the case of ctenophores and other marine organisms. Coupled with transcriptome analysis, and immunohistochemical studies of receptor expression in vivo, experiments with heterologous expression systems can provide valuable insight into cellular activity, prior to more challenging functional studies on native tissues.

STAR-RIS aided Secure Communication for Endogenous Wiretap Broadcast Channels with Two Users

STAR-RIS aided Secure Communication for Endogenous Wiretap Broadcast Channels with Two Users

Two-pore channel-2 and inositol trisphosphate receptors coordinate Ca2+ signals between lysosomes and the endoplasmic reticulum

Lysosomes and the endoplasmic reticulum (ER) are Ca2+ stores mobilized by the second messengers NAADP and IP3, respectively. Here, we establish Ca2+ signals between the two sources as fundamental building blocks that couple local release to global changes in Ca2+. Cell-wide Ca2+ signals evoked by activation of endogenous NAADP-sensitive channels on lysosomes comprise both local and global components and exhibit a major dependence on ER Ca2+ despite their lysosomal origin. Knockout of ER IP3 receptor channels delays these signals, whereas expression of lysosomal TPC2 channels accelerates them. High-resolution Ca2+ imaging reveals elementary events upon TPC2 opening and signals coupled to IP3 receptors. Biasing TPC2 activation to a Ca2+-permeable state sensitizes local Ca2+ signals to IP3. This increases the potency of a physiological agonist to evoke global Ca2+ signals and activate a downstream target. Our data provide a conceptual framework to understand how Ca2+ release from physically separated stores is coordinated.

Trifunctional Saxitoxin Conjugates for Covalent Labeling of Voltage-Gated Sodium Channels.

Voltage-gated sodium ion channels (NaV s) are integral membrane protein complexes responsible for electrical signal conduction in excitable cells. Methods that enable selective labeling of NaV s hold potential value for understanding how channel regulation and post-translational modification are influenced during development and in response to diseases and disorders of the nervous system. We have developed chemical reagents patterned after (+)-saxitoxin (STX) - a potent and reversible inhibitor of multiple NaV isoforms - and affixed with a reactive electrophile and either a biotin cofactor, fluorophore, or 'click' functional group for labeling wild-type channels. Our studies reveal enigmatic structural effects of the probes on the potency and efficiency of covalent protein modification. Among the compounds analyzed, a STX-maleimide-coumarin derivative is most effective at irreversibly blocking Na+ conductance when applied to recombinant NaV s and endogenous channels expressed in hippocampal neurons. Mechanistic analysis supports the conclusion that high-affinity toxin binding is a prerequisite for covalent protein modification. Results from these studies are guiding the development of next-generation tool compounds for selective modification of NaV s expressed in the plasma membranes of cells.

Sodium channel endocytosis drives axon initial segment plasticity.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

Dehydroepiandrosterone sulfate (DHEAS) is an endogenous Kv7 channel modulator that reduces Kv7/M-current suppression and inflammatory pains.

Neuronal Kv7 voltage-gate potassium channels generate the M-current and regulate neuronal excitability. Here, we report that dehydroepiandrosterone sulfate (DHEAS) is an endogenous Kv7 channel modulator that attenuates Gq-coupled receptor induced M-current suppression. DHEAS reduced muscarinic agonist-induced Kv7-current suppression of either Kv7.1, Kv7.2, Kv7.4 or Kv7.5 homomeric currents and endogenous M-currents in rat sympathetic ganglion neurons. However, DHEAS per se did not alter the voltage-dependence of these Kv7 homomeric channels or the m1 receptor-induced activation of phospholipase C or protein kinase C. DHEAS treated Kv7.2 homomeric currents became resistant to depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by voltage-activated phosphatase, Ci-VSP or eVSP. Our computational models predicted a novel binding site for DHEAS in the cytoplasmic domain of Kv7 subunits. A single point mutation of the predicted key histidine into cysteine in the rat Kv7.2 subunit, rKv7.2(H558C), resulted in a loss of effects of DHEAS on muscarinic Kv7 current suppression. Furthermore, in vivo administration of DHEAS in mice of both sexes reduced late phase pain responses in the formalin paw test. However, it did not have effects on early phase responses in the formalin paw test or responses in the hot plate test. Co-administration of a selective Kv7 inhibitor, XE991, and DHEAS eliminated analgesic effects of DHEAS in late phase responses in the formalin paw test. Collectively, these results suggest that DHEAS attenuates M-current suppression by stabilizing PIP2-Kv7 subunit interaction and can mitigate inflammatory pain.Significance StatementM-current suppression induced by stimulation of Gq-coupled receptors is a form of Kv7 current modulation that can reversibly increase neuronal excitability. This study demonstrates that DHEAS, an endogenous steroid hormone, is a novel Kv7 channel modulator that can attenuate M-current suppression without affecting basal Kv7 channel kinetics. Administration of DHEAS in vivo alleviated inflammatory pain in rodents. These results suggest that the degree of M-current suppression can be dynamically regulated by small molecules. Therefore, this novel form of Kv7 channel regulation holds promising potential as a therapeutic target for sensitized nervous activities such as inflammatory pain.