Potassium Channels Research Articles

Variability in flow-induced vasodilation mechanisms in cerebral arteries: the impact of different hyperbaric oxygen protocols.

The present study aimed to assess the mechanisms of flow-induced dilation (FID) altered by acute/intermittent hyperbaric oxygenation (HBO2) in isolated middle cerebral arteries of healthy male Sprague-Dawley rats (n = 96) and randomized to the Ac-HBO2 group (exposed to a single HBO2 session, 120 minutes of 100% O2 at 2.0 bars), the 4Dys-HBO2 group (4 consecutive days of single HBO2 sessions, analyzed on the fifth day), and the CTRL (untreated) group. Results demonstrated increased vascular oxidative stress and decreased vascular nitric oxide bioavailability, as measured by direct fluorescence microscopy, leading to attenuated FID in the Ac-HBO2 group compared with the CTRL and 4Dys-HBO2 groups. Superoxide scavenging restored FID. Moreover, the increased expression of antioxidative enzymes in the cerebral vasculature in the 4Dys-HBO2 group indicates the ability of intermittent HBO2 to activate antioxidative mechanisms. Importantly, the results suggest a switch or at least activation of the compensatory mechanism of FID after HBO2 from nitric oxide-dependent to epoxygenase metabolite-mediated via TRPV4 (transient receptor potential cation channel subfamily V member 4) and potassium channels, as demonstrated by increased protein expression of KCNMB1 (potassium calcium-activated channel subfamily M regulatory beta subunit 1), TRPV4, and Kir2 (a component of the inward rectifier-type potassium channel Kir2) in the vasculature. Overall, acute HBO2 modulates FID in cerebral vessels by increasing oxidative stress and altering the subsequent mechanisms of FID, which are mainly mediated by nitric oxide, while suppressing potassium and TRPV4 channel function/expression due to increased oxidative stress. Moreover, intermittent HBO2 activates antioxidative mechanisms and the compensatory mechanism of FID from nitric oxide-dependent to epoxygenase metabolite-mediated mechanisms via TRPV4, KCNMB1 and Kir2.1.

Shaker/Kv1 potassium channel SHK-1 protects against pathogen infection and oxidative stress in C. elegans.

The Shaker/Kv1 subfamily of voltage-gated potassium (K+) channels is essential for modulating membrane excitability. Their loss results in prolonged depolarization and excessive calcium influx. These channels have also been implicated in a variety of other cellular processes, but the underlying mechanisms remain poorly understood. Through comprehensive screening of K+ channel mutants in C. elegans, we discovered that shk-1 mutants are highly susceptible to bacterial pathogen infection and oxidative stress. This vulnerability is associated with reduced glycogen levels and substantial mitochondrial dysfunction, including decreased ATP production and dysregulated mitochondrial membrane potential under stress conditions. SHK-1 is predominantly expressed and functions in body wall muscle to maintain glycogen storage and mitochondrial homeostasis. RNA-sequencing data reveal that shk-1 mutants have decreased expression of a set of cation-transporting ATPases (CATP), which are crucial for maintaining electrochemical gradients. Intriguingly, overexpressing catp-3, but not other catp genes, restores the depolarization of mitochondrial membrane potential under stress and enhances stress tolerance in shk-1 mutants. This finding suggests that increased catp-3 levels may help restore electrochemical gradients disrupted by shk-1 deficiency, thereby rescuing the phenotypes observed in shk-1 mutants. Overall, our findings highlight a critical role for SHK-1 in maintaining stress tolerance by regulating glycogen storage, mitochondrial homeostasis, and gene expression. They also provide insights into how Shaker/Kv1 channels participate in a broad range of cellular processes.

The roles of melatonin and potassium channels in relaxation response to ang 1–7 in diabetic rat isolated aorta

The roles of melatonin and potassium channels in relaxation response to ang 1–7 in diabetic rat isolated aorta

Microbial metabolites tune amygdala neuronal hyperexcitability and anxiety-linked behaviors.

Changes in gut microbiota composition have been linked to anxiety behavior in rodents. However, the underlying neural circuitry linking microbiota and their metabolites to anxiety behavior remains unknown. Using male C57BL/6J germ-free (GF) mice, not exposed to live microbes, increased anxiety-related behavior was observed correlating with a significant increase in the immediate early c-Fos gene in the basolateral amygdala (BLA). This phenomenon coincided with increased intrinsic excitability and spontaneous synaptic activity of BLA pyramidal neurons associated with reduced small conductance calcium-activated potassium (SK) channel currents. Importantly, colonizing GF mice to live microbes or the microbial-derived metabolite indoles reverted SK channel activities in BLA pyramidal neurons and reduced the anxiety behavioral phenotype. These results are consistent with a molecular mechanism by which microbes and or microbial-derived indoles, regulate functional changes in the BLA neurons. Moreover, this microbe metabolite regulation of anxiety links these results to ancient evolutionarily conserved defense mechanisms associated with anxiety-related behaviors in mammals.

Antihypertensive effect of sinapine extracted from rapeseed meal in 2K1C hypertensive rats

To extract sinapine from rapeseed meal and investigate its antihypertensive function and mechanism. Blood pressure was measured before and after sinapine administration to evaluate sinapine’s immediate antihypertensive function. Twokidney, oneclip (2K1C) hypertensive rats were given sinapine for four weeks, with weekly blood pressure monitoring. The renin angiotensin aldosterone system (RAAS), including the levels of renin, angiotensin I (Ang I), angiotensin II (Ang II), aldosterone (ALD), angiotensin-converting enzyme (ACE) and other molecules related to blood pressure, such as NO, prostacyclin (PGI2), endothelin-1 (ET1), and thromboxane A2 (TXA2), were measured in rat blood. The impact of sinapine on vascular endothelial cell (A10) calcium and potassium channels was assessed using the patch-clamp technique. One-time or long-term administration of sinapine significantly reduced the rats’ systolic blood pressure (SBP), diastolic pressure (DBP), and mean blood pressure (MBP). Sinapine also decreased the levels of Ang II and ALD. Furthermore, sinapine effectively inhibited ACE activation, increased NO levels, and blocked L-type calcium channels. Sinapine has an antihypertensive function and achieves this process through multiple targets.

Inhibition of BK channels by GABAb receptors enhances intrinsic excitability of layer 2/3 vasoactive intestinal polypeptide-expressing interneurons in mouse neocortex.

GABAb receptors (GABAbRs) affect many signalling pathways, and hence the net effect of the activity of these receptors depends upon the specific ion channels that they are linked to, leading to different effects on specific neuronal populations. Typically, GABAbRs suppress neuronal activity in the cerebral cortex. Previously, we found that neocortical parvalbumin-expressing cells are strongly inhibited through GABAbRs, whereas somatostatin interneurons are immune to this modulation. Here, we employed in vitro whole-cell patch-clamp recordings to study whether GABAbRs modulate the activity of vasoactive intestinal polypeptide-expressing interneurons (VIP-INs) in layer (L) 2/3 of the mouse primary somatosensory cortex. Utilizing machine learning algorithms (hierarchical clustering and principal component analysis), we revealed that one VIP-IN cluster (about 68% of all VIP-INs) was sensitive to GABAbR activation. Paradoxically, when recordings were performed in standard conditions with high extracellular Ca2+ level, GABAbRs indirectly inhibited the activity of large conductance voltage- and calcium-activated potassium (BK) channels and reduced GABAaR-mediated inhibition, leading to an increase in intrinsic excitability of these interneurons. However, a classical inhibitory effect of GABAbRs on L2/3 VIP-INs was observed in modified artificial cerebrospinal fluid with physiological (low) Ca2+ concentration. Our results are essential for a deeper understanding of mechanisms underlying the modulation of cortical networks. KEY POINTS: Layer 2/3 vasoactive intestinal polypeptide-expressing interneurons (VIP-INs) in the mouse somatosensory cortex cluster into three electrophysiological types differentially sensitive to GABAb receptors (GABAbRs). The majority of VIP-INs (type 1, about 68% of all VIP-INs) are regulated through pre- and postsynaptic GABAbRs, while a subset of these interneurons (types 2 and 3) is controlled only presynaptically. The net effect of GABAbR activation on VIP-IN excitability depends on [Ca2+] in artificial cerebrospinal fluid. When [Ca2+] is high (2.5mM), GABAbRs indirectly inhibit BK channels and reduce GABAaR inhibition leading to increased intrinsic excitability of type 1 VIP-INs. When [Ca2+] is low (1mM), which is more physiological, BK channels do not regulate the intrinsic excitability of VIP-INs and thus postsynaptic GABAbRs canonically decrease the intrinsic excitability of type 1 VIP-INs.

Reduced GIRK expression in midbrain dopamine neurons during prolonged abstinence from fentanyl self-administration.

Despite decades of research and medical development, relapse to drug seeking continues to be a significant challenge in the treatment of substance use disorders. GABAB receptor (GABAB-R) agonists have been shown preclinically to inhibit relapse by acting on midbrain dopamine (DA) neurons and are sometimes used off-label for the treatment of alcohol use disorder. Studies in rodent models show reduced GABAB-R signaling in DA neurons after exposure to stimulants. Similarly, our recent data demonstrated reduced GABAB-R currents in DA neurons during prolonged abstinence from fentanyl vapor self-administration (SA). However, the mechanism of opioid-induced changes in GABAB-R currents is not well understood. In addition, GABAB-R agonists are plagued with a plethora of side effects limiting their potential clinical use. In this study we aimed to answer the following questions: first, can we use GABAB-R positive allosteric modulators (PAMs) to inhibit relapse to opioid seeking? Secondly, how do opioids result in reduced GABAB-R signaling during prolonged abstinence? To this end, we tested the effects of a novel GABAB-R PAM (KK-92A) on reinstatement of drug seeking in a rat model of intravenous (IV) fentanyl SA. Using in situ hybridization with RNAscope, we examined the effects of opioids on mRNA levels of various genes involved in GABAB-R signaling, in two rodent models of opioid addiction including a rat model of IV fentanyl SA and a mouse model of fentanyl vapor SA. Our results show that KK-92A inhibits relapse to fentanyl but not sucrose-seeking in rats, and fentanyl SA results in reduced mRNA levels of the G protein-coupled inwardly rectifying potassium channel subtypes 2 and 3 (GIRK2/3). These findings suggest that PAMs like KK-92A are a potential therapeutic strategy for opioid use disorder and their effect is likely due to rectifying GABAB-R mediated inhibition of midbrain DA neurons, which is reduced after opioid SA due to reduced GIRK2/3 expression.

Targeting the hERG1/β1 integrin complex in lipid rafts potentiates statins anti-cancer activity in pancreatic cancer

Plasma membrane macromolecular complexes function as signaling hubs that regulate cell behavior, which is particularly relevant in cancer. Our study provides evidence that the complex formed by the hERG1 potassium channel and the β1 subunit of integrin receptors preferentially localizes in Lipid Rafts (LRs) in Pancreatic Ductal Adenocarcinoma (PDAC) cell lines and primary samples. The complex recruits the p85 subunit of phosphatidyl-inositol-3-kinase (PI3K), activating phosphoinositide metabolism and triggering an intracellular signaling pathway centered on Akt. This pathway ultimately affects cancer cell proliferation through cyclins and p21, and cell migration through the small GTPase Rac-1 and f-actin organization. The hERG1/β1 integrin complex in LRs can be dissociated and the downstream signaling pathway can be inhibited by either disrupting LRs through methyl-beta-cyclodextrin (MβCD) or inhibiting cholesterol synthesis by statins. Treatment with a single chain bispecific antibody—scDb-hERG1-β1—specifically targeting the complex significantly potentiates the effects of both MβCD and statins on intracellular signaling. Consequently, these treatments decrease PDAC cell proliferation and motility in vitro. From a pharmacological perspective, different statins produce anti-neoplastic effects in synergy with scDb-hERG1-β1. Such combination also enhances tumor sensitivity to chemotherapeutic drugs, such as gemcitabine and oxaliplatin. The efficacy of these combination treatments depends on the amount of the hERG1/β1 integrin complex present on the plasma membrane of cancer cells. Finally, the combined treatment with statins and scDb-hERG1-β1 significantly reduces tumor growth and improves survival in vivo, in a preclinical mouse model. These results suggest that the combination of scDb-hERG1-β1 and statins represent a potential novel strategy for treating PDAC patients.

Regional Alterations in Müller Cell Protein Expression in Human and a Rat Model of Geographic Atrophy.

Despite being crucial to neuronal survival, the role Müller cells play in geographic atrophy (GA) has only recently been considered. We investigated whether Müller cells retain their normal functional profile or form a fibrotic scar when remodeling in human GA eyes and our subretinal sodium iodate (NaIO3) model. Sprague Dawley rats given subretinal injections of NaIO3 (5 mg/mL) were sacrificed at 3 and 12 weeks. Cryosections and retinal flatmounts from rats and cryosections from human GA eyes were stained with antibodies against the Müller cell proteins glutamine synthetase (GS), inwardly rectifying potassium channel 4.1 (Kir4.1), aquaporin 4 (AQP4), cellular retinaldehyde-binding protein 1 (CRALBP), and glial fibrillary acidic protein (GFAP), as well as alpha smooth muscle actin (α-SMA), fibronectin, and collagens I and IV. The immunofluorescence intensity of AQP4 and Kir4.1 was quantified using Image J, and Kir4.1 protein levels were verified by western blot. In both human GA eyes and NaIO3-injected rats, Müller cell processes at the external limiting membrane (ELM) descent and in the subretinal membrane exhibited increased GS expression. GFAP was elevated throughout the Müller cells. AQP4 staining at the ELM descent was particularly pronounced throughout the radial processes, including those extending into the subretinal space. In NaIO3-injected rats, perivascular Kir4.1 expression significantly decreased in the atrophic retina, but expression increased in the subretinal glial membrane. α-SMA and extracellular matrix proteins were not detected in the subretinal membrane. Our findings underscore the persistence of homeostatic proteins, albeit altered, in Müller cells as they remodel and extend into the subretinal space.

Functions and clinical significance of KCNB1 in esophageal squamous cell carcinoma.

Voltage-gated potassium channel subfamily B member 1 (KCNB1) encodes the α-subunit of the Kv2.1 channel and mediates transmembrane potassium transport. The functions and mechanisms underlying KCNB1 activation have been examined in various cancer types; however, its role in esophageal squamous cell carcinoma (ESCC) remains unclear. Therefore, the present study investigated the involvement of KCNB1 in tumor progression and the clinicopathological significance of its expression in ESCC. Knockdown experiments using KCNB1 small interfering RNA were performed on the human ESCC cell lines, KYSE70 and TE5, and changes in cell proliferation, the cell cycle, apoptosis, migration, and invasion were assessed. Gene expression profiles were examined using a microarray analysis. An immunohistochemical (IHC) analysis was performed on 129 primary tumor samples from ESCC patients who underwent curative esophagectomy. Cell proliferation, G2-M phase progression, migration, and invasion were inhibited, and apoptosis was induced in KCNB1-depleted cells. Microarray results showed that KCNB1 gene expression affected Ephrin receptor signaling by suppressing EPHB1, EPHB2, and ERK1/2 gene expression. IHC results revealed a relationship between high KCNB1 expression and a poor prognosis. High KCNB1 expression was extracted as an independent prognostic factor in a multivariate analysis of 5-year relapse-free survival in ESCC patients (p = 0.0197). Cell proliferation is controlled by KCNB1 through its regulation of ERK1/2 gene expression via ephrin receptor signaling. A relationship was observed between KCNB1 and the prognosis of ESCC patients, indicating its potential as a biomarker for cancer progression and in targeted therapy for ESCC.

Immunomodulatory effect of selective COX-2 inhibitor celecoxib on the neuropathological disorders and immunoinflammatory response induced by Kaliotoxin from Androctonus australis venom.

The immune response is increasingly being linked to the pathogenic processes underlying neurological disorders including potassium channel malfunction. Few investigations, meanwhile, have shown how cyclooxygenase-2 (COX-2) is involved in the neuroimmunopathology linked to potassium channel failure. Thus, using an animal model of neuropathology caused by kaliotoxin, an exclusive blocker of voltage-gated potassium channels from the scorpion venom of Androctonus australis hector, we examined the immunomodulatory impact of celecoxib (selective inhibitor of COX-2). The neural and systemic pathogenic effects of KTX can be considerably reduced by celecoxib-mediated COX-2 inhibition, according to the results. It most certainly works via controlling the immunoinflammatory exposure by raising IL-10 levels; decreasing proinflammatory cytokine levels including mostly TNFα and IL-6, and balancing oxidative status. Along with that, by significantly promoting tissue healing, COX-2 inhibitor also enhances cellular metabolism. One potential treatment approach for immunoinflammatory exacerbations linked to neurodegenerative is the COX-2 inhibitor.

Pharmacodynamic Elucidation of Pelargonium zonale (L.) L'Hér. ex Aiton for its Folkloric Claims in Diarrhea and Asthma via in Vitro, in Vivo and in Silico Methods

Ethnopharmacological relevance Pelargonium zonale (L.) L'Hér. ex Aiton ( P.zonale) is used against various cardiovascular, gastrointestinal and respiratory diseases involving smooth muscles relaxation. It is traditionally used for the treatment of diarrhea, gastropathy, and toothache. Aim of study The current study was designed to validate its conventional applications, we conducted the investigational studies to evaluate the potential mode of operation in the management of disorders pertaining to the cardiovascular system, respiratory tract, and gastrointestinal tract via in-vitro, in-vivo and in-silico studies. Material and methods The bioactive compounds were identified via HPLC done by using a binary-gradient solvent system containing of C-18 column (250 × 4.6 mm, 5 µm), flow rate of 0.8 ml/min, and run time of 36 min was used for the illumination of P.zonale crude extract chromatogram. These compounds were predicted spasmolytic and bronchodilation potential in molecular docking. Invitro methods confirmed the spasmolytic and bronchodilator effects. Analgesic, anti-inflammatory and antidiarrheal experiments were conducted in in-vivo studies. Results Experimental investigations have disclosed that it possesses antidiarrheal, anti-inflammatory, and analgesic attributes. In vitro examinations of this botanical specimen demonstrated its spasmolytic, vasorelaxant, ionotropic, and bronchodilator activities, which involve adrenergic, muscarinic, and potassium ion channels by alleviating sustained contractions induced by phenylephrine (1 µM), carbamylcholine (1 µM), and KCl (25 mM L−1) respectively. The properties associated with the opening of potassium channels were validated by conducting repeat experiments in the presence of tetraethylammonium (TEA) and glibenclamide (GB), which confirmed the activation of voltage-activated and ATP-sensitive potassium channels. Both in vitro and in vivo investigations reveal the existence of significant smooth muscle relaxant activity, further corroborated by in silico studies. Conclusion In vitro and in vivo studies reveal substantial capacities for smooth muscle relaxation, which encompass bronchodilation, spasmolytic activity, and vasodilation, with these observations being corroborated by molecular docking analyses. However, further studies are needed to define the clinical significance and safety treatment with P.zonale.

Pore blocking mechanisms of centipede toxin SsTx-4 on the inwardly rectifying potassium channels.

The peptide toxin SsTx-4 derived from venom of centipede Scolopendra subspinipes mutilans was characterized as a potent antagonist of the inwardly rectifying potassium (Kir) channel subtypes Kir1.1, Kir4.1, and Kir6.2 in our previous study. Alanine-scanning mutagenesis analysis identified key molecular determinants on the SsTx-4 toxin interacting with these Kir channels, as well as those on the Kir6.2 channel interacting with the toxin. However, the key residues on Kir1.1 and Kir4.1 channels responsible for binding SsTx-4 remain unclear. Here, using a combination of site-directed mutagenesis, patch-clamp analysis, molecular docking with AlphaFold 3, and molecular dynamic simulations, we revealed that SsTx-4 acted on the Kir channels as a pore blocker, with K13 on toxin serving as the functional pore-blocking residue and other residues on it contributing to stabilize the toxin-channel complex by binding to multiple residues on the wall of the channels' outer vestibule, involving E104 on Kir1.1; D100, L115, and F133 on Kir4.1; and E108, S113, H115, and M137 on Kir6.2. Collectively, these findings advanced our understanding on the interaction between Kir channels and this prototype Kir antagonist, providing insights that could inspire the development of more potent and specific Kir subtype blockers in the future.

Unraveling pH Regulation of TMEM175, an Endolysosomal Cation Channel With a Role in Parkinson's Disease.

Transmembrane protein 175 (TMEM175) is an endolysosomal cation channel, which has attracted much attention recently from academics and the pharmaceutical industry alike since human mutations in TMEM175 were found to be associated with the development of Parkinson's disease (PD). Thus, gain-of-function mutations were identified, which reduce and loss-of-function mutations, which increase the risk of developing PD. After having been characterized as an endolysosomal potassium channel initially, soon after TMEM175 was claimed to act as a proton channel. In fact, recent evidence suggests that depending on the conditions, TMEM175 can act as either a potassium or proton channel, without acting as an antiporter or exchanger. A recent work has now identified amino acid H57 to be directly involved in gating, increasing proton conductance of the channel while leaving the potassium conductance unaffected. We review here the current knowledge of TMEM175 function, pharmacology, physiology, and pathophysiology. We discuss the potential of this ion channel as a novel drug target for the treatment of neurodegenerative diseases such as PD, and we discuss the discovery of H57 as proton sensor.

S-Denitrosylation counteracts local inflammation and improves survival in mice infected with K. pneumoniae.

Sepsis and septic shock remain are significant causes of mortality in the world. The inflammatory response should be at the basis of all organ dysfunction such as cardiovascular dysfunction, characterized by severe hypotension refractory to volume replacement and vasoconstrictor therapy. Nitric oxide (NO) has been implicated as a key element in both inflammatory and cardiovascular components of sepsis. In addition to activating soluble guanylate cyclase and potassium channels, NO also modifies proteins post-translationally by reacting with protein thiol groups, yielding S-nitrosothiols (RS-NO), which can act as endogenous NO reservoirs. Besides its use in quantifying free sulfhydryl groups of proteins and non-protein thiols, DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] has also been used as a pharmacological tool due to its specificity for oxidizing reactive sulfhydryl groups. Here we aimed to investigate the effects of DTNB in the inflammatory aspects of a sepsis model and to verify whether its effects can be attributed to S-denitrosylation. Anesthetized female Swiss mice were intratracheally injected with 1×108 CFU of K. pneumoniae. Twelve hours after pneumonia-induced sepsis, the animals were injected with vehicle (sodium bicarbonate 5%, s.c.) or DTNB (31.5, 63 and 126 μmol/kg, s.c.). Twenty-four hours post-sepsis induction, plasma, bronchoalveolar lavage (BAL), and lung tissues were collected for assays (protein, cell count, nitrite+nitrate levels (NOx), cytokine levels, and sulfhydryl groups). In addition, lung S-nitrosylated proteins were visualized by a modified tissue assay for S-nitrosothiols. Sepsis induced a significant vascular leakage in the lungs and elevated NOx levels in BAL, both reduced by DTNB. BAL leukocytosis and elevated IL-1β induced by sepsis were also reduced by DTNB, whereas it did not affect bacterial dissemination to liver, heart and BAL. Sepsis reduced free sulfhydryl groups in BAL and lung and DTNB did not change it. On the other hand, DTNB substantially reduced protein S-nitrosylation levels in the lung parenchyma and halved sepsis-induced mortality in septic mice. Our results show that the administration of DTNB 12 h after bacterial instillation reduced most of the local inflammatory parameters and, more importantly, decreased mortality. These beneficial effects may be due to S-denitrosylation of RS-NO pools carried out by DTNB. Since DTNB was effective in reducing the inflammatory process after its onset, this mechanism of action could serve as a valuable proof of concept for compounds that can be useful to interfere with sepsis outcome.

Insight into a multifunctional potassium channel Kv1.3 and its novel implication in chronic kidney disease.

Chronic kidney disease (CKD), a global public health problem, causes substantial morbidity and mortality worldwide. Innovative therapeutic strategies to mitigate the progression of CKD are needed due to the limitations of existing treatments. Kv1.3, a voltage-gated potassium ion channel, plays a crucial role in multiple biological processes, including cell proliferation, apoptosis, energy homeostasis, and migration. Inhibition of the Kv1.3 channels has shown beneficial effects in the therapy of a wide range of human diseases such as cancer, autoimmune and neuroinflammatory diseases. Increasing evidence reveals a close link between Kv1.3 and CKD. This review summarises the most recent insights into the physiological functions of the Kv1.3 channel and its pharmacological modulators. Furthermore, the therapeutic potential of targeting Kv1.3 for CKD is also discussed. Collectively, these studies suggested that Kv1.3 channels may serve as a novel target for CKD therapy.

Atp1a2 and Kcnj9 Are Candidate Genes Underlying Sensitivity to Oxycodone-Induced Locomotor Activation and Withdrawal-Induced Anxiety-Like Behaviors in C57BL/6 Substrains.

Opioid use disorder is heritable, yet its genetic etiology is largely unknown. C57BL/6J and C57BL/6NJ mouse substrains exhibit phenotypic diversity in the context of limited genetic diversity which together can facilitate genetic discovery. Here, we found C57BL/6NJ mice were less sensitive to oxycodone (OXY)-induced locomotor activation versus C57BL/6J mice in a conditioned place preference paradigm. Narrow-sense heritability of OXY-induced locomotor activity traits ranged from 0.22 to 0.31, implicating suitability for genetic analysis. Quantitative trait locus (QTL) mapping in an F2 cross identified a chromosome 1 QTL explaining 7%-12% of the variance in OXY locomotion and anxiety-like withdrawal in the elevated plus maze. A second QTL for EPM withdrawal behavior on chromosome 5 near Gabra2 (alpha-2 subunit of GABA-A receptor) explained 9% of the variance. To narrow the chromosome 1 locus, we generated recombinant lines spanning 163-181 Mb, captured the QTL for OXY locomotor traits and withdrawal, and fine-mapped a 2.45-Mb region (170.16-172.61 Mb). Transcriptome analysis identified five, localized striatal cis-eQTL transcripts and two were confirmed at the protein level (KCNJ9, ATP1A2). Kcnj9 codes for a potassium channel (GIRK3) that is a major effector of mu opioid receptor signaling. Atp1a2 codes for a subunit of a Na+/K+ ATPase enzyme that regulates neuronal excitability and shows functional adaptations following chronic opioid administration. To summarize, we identified two candidate genes underlying the physiological and behavioral properties of opioids, with direct preclinical relevance to investigators employing these widely used substrains and clinical relevance to human genetic studies of opioid use disorder.

Kir5.1 regulates Kir4.2 expression and is a key component of the 50-pS inwardly rectifying potassium channel in basolateral membrane of mouse proximal tubules.

Kir5.1 encoded by Kcnj16 is an inwardly rectifying K+ channel subunit, and it possibly interacts with Kir4.2 subunit encoded by Kcnj15 for assembling a Kir4.2/Kir5.1 heterotetramer in the basolateral membrane of mouse proximal tubule. We now used patch clamp technique to examine basolateral K+ channels of mouse proximal tubule (PT) and an immunoblotting/immunofluorescence (IF) staining microscope to examine Kir4.2 expression in wild-type and Kir5.1-knockout mice. IF staining shows that Kir4.2 was exclusively expressed in the proximal tubule, whereas Kir5.1 was expressed in the proximal tubule and distal nephrons including distal convoluted tubule. Immunoblotting showed that the expression of Kir4.2 monomer was lower in Kir5.1-knockout mice than that in the wild-type mice. In contrast, Kir4.1 monomer expression was increased in Kir5.1 knockout mice. IF images further demonstrated that the basolateral membrane staining of Kir4.2 was significantly decreased in Kir5.1 knockout mice. This is in sharp contrast to Kir4.1, which also interacts with Kir5.1 in the distal nephron, and IF images show that Kir4.1 membrane expression was still visible and unchanged in Kir5.1 knockout mice. The single channel recording detected a 50-pS inwardly rectifying K+ channel, presumably a Kir4.2/Kir5.1 heterotetramer, in the basolateral membrane of the proximal tubule of Kir5.1 wild-type mice. However, this 50-pS K+ channel was completely absent in the basolateral membrane of the proximal tubule of Kir5.1 knockout mice. Moreover, the membrane potential of the proximal tubule was less negative in Kir5.1 knockout mice than wild-type mice. We conclude that Kir5.1 is essential for assembling basolateral 50-pS K+ channel in proximal tubule and that deletion of Kir5.1 decreased Kir4.2 expression in the proximal tubule thereby decreasing the basolateral K+ conductance and the membrane potentials.NEW & NOTEWORTHY Our studyprovides direct evidence for the notion that Kir5.1 is a key component of a 50-60 pS inwardly-rectifying-K+ channel, a main type K+ channel in the basolateral-membrane of PT. Also, we demonstrate that deletion of Kir5.1 decreased Kir4.2 protein expression including the basolateral-membrane in PT. Finally, depolarization ofPT-membrane- potential in Kir5.1-knockout mice suggests that Kir4.2 alone is not able to sustain basolateral K+ conductance of the PT in the absence of Kir5.1.

Abstract TMP75: Reduced thrombus immunoreactivity is transcriptionally associated with greater NIHSS severity at presentation of ischemic stroke: Analysis from the INSIGHT registry

Funding: The INSIGHT Registry is funded by Penumbra. Introduction: Thrombi have shown to modify and be modified by their microenvironment throughout the pathogenesis of ischemic stroke. The molecular mechanisms by which this occurs and how it corresponds to symptomatic presentation is unknown. Here, we identify a transcriptomic signature in thrombi associated with increased NIHSS severity at presentation, suggesting a mechanistic role for immune clearance in clinical severity of ischemic stroke. Methods: The INSIGHT registry is a prospective, multicenter, multi-omic registry focused on elucidating the molecular underpinnings of stroke from analysis of clot and interarterial blood. RNA sequencing of 10,990 genes from 292 thrombi from patients undergoing thrombectomy was aligned, normalized, and residualized using Rsubread, edgeR, sva, and limma/voom. Signed co-expression modules were constructed using WGCNA, and each module eigengene (a measure of a module’s principal component) was correlated with NIHSS at presentation using Pearson’s correlation. Networks were visualized and analyzed in cytoscape. Hub genes were determined by number of undirected edges. Network enrichments were conducted using Gene Ontology biological processes and cellular components. Results: The mean NIHSS in this cohort was 15.14± 7.45. 3 co-expression modules out of 26 total modules detected in the data significantly inversely correlated with NIHSS at presentation. These networks corresponded to downregulation of neuronal activation (p=0.042), interferon signaling (p=0.039), and T cell activation (p=0.023), respectively. Major hub genes in each module included KCNAB2, a potassium channel associated with epilepsy and implicated in susceptibility to poor stroke outcomes in aged mice brains, SRSF11, associated with cognitive decline, PNISR, associated with interferon signaling, and ZAP70, associated with T-cell differentiation. Conclusion: The association of immune downregulation with increased NIHSS severity suggests that robust immune clearance may mitigate poor outcomes in ischemic stroke, and that thrombi-mediated modification of the local immune environment may exacerbate symptomatic presentation. Conversely, the association of major hub genes with known cognitive decline suggests that thrombi are modified by the neurovascular unit and neurons of surrounding tissues and post-thrombectomy sequencing may provide insight into the underlying health of the surrounding parenchyma.

GDF10 promotes rodent cardiomyocyte maturation during the postnatal period.

Cardiomyocytes and cardiac fibroblasts undergo coordinated maturation after birth, and cardiac fibroblasts are required for postnatal cardiomyocyte maturation in mice. Here, we investigate the role of cardiac fibroblast-expressed Growth Differentiation Factor 10 (GDF10) in postnatal heart development. In neonatal mice, Gdf10 is expressed specifically in cardiac fibroblasts, with its highest expression coincident with the onset of cardiomyocyte cell cycle arrest and transition to hypertrophic growth. In neonatal rat ventricular myocyte (NRVM) cultures, GDF10 treatment promotes cardiomyocyte maturation indicated by increased binucleation, downregulation of cell cycle progression genes, and upregulation of cell cycle inhibitor genes. GDF10 treatment leads to an increase in cardiomyocyte cell size, together with increased expression of mature sarcomeric protein isoforms and decreased expression of fetal cardiac genes. RNAsequencing of GDF10-treated NRVM shows an increase in the expression of genes related to myocardial maturation, including upregulation of sodium and potassium channel genes. In vivo, loss of Gdf10 leads to a delay in myocardial maturation indicated by decreased cardiomyocyte cell size and binucleation, as well as increased mitotic activity, at postnatal (P) day 7. Further, induction of mature sarcomeric protein isoform gene expression is delayed, and expression of cell cycle progression genes is prolonged. However, by P10, indicators of cardiomyocyte maturation and mitotic activity are normalized in Gdf10-null hearts relative to controls. Together, these results implicate GDF10 as a novel crosstalk mediator between cardiomyocytes and cardiac fibroblasts, which is required for appropriate timing of cardiomyocyte maturation steps including binucleation, hypertrophy, mature sarcomeric isoform gene expression, and cell cycle arrest in the postnatal period.