Kir Channels Research Articles

Development of new Kir2.1 channel openers from propafenone analogues.

Reduced inward rectifier potassium channel (Kir2.1) functioning is associated with heart failure and may cause Andersen-Tawil Syndrome, among others characterized by ventricular arrhythmias. Most heart failure or Andersen-Tawil Syndrome patients are treated with β-adrenoceptor antagonists (β-blockers) or sodium channel blockers; however, these do not specifically address the inward rectifier current (IK1) nor aim to improve resting membrane potential stability. Consequently, additional pharmacotherapy for heart failure and Andersen-Tawil Syndrome treatment would be highly desirable. Acute propafenone treatment at low concentrations enhances IK1 current, but it also exerts many off-target effects. Therefore, discovering and exploring new IK1-channel openers is necessary. Effects of propafenone and 10 additional propafenone analogues were analysed. Currents were measured by single-cell patch-clamp electrophysiology. Kir2.1 protein expression levels were determined by western blot analysis and action potential characteristics were further validated in human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMCs). Molecular docking was performed to obtain detailed information on drug-channel interactions. Analogues GPV0019, GPV0057 and GPV0576 strongly increased the outward component of IK1 while not affecting the Kir2.1 channel expression levels. GPV0057 did not block IKr at concentrations below 0.5μmol L-1 nor NaV1.5 current below 1μmol L-1. Moreover, hiPSC-CMC action potential duration was also not affected by GPV0057 at 0.5 and 1μmol L-1. Structure analysis indicates a mechanism by which GPV0057 might enhance Kir2.1 channel activation. GPV0057 has a strong efficiency towards increasing IK1, which makes it a good candidate to address IK1 deficiency-associated diseases.

Chronic ethanol exposure in mice evokes pre- and postsynaptic deficits in GABAergic transmission in ventral tegmental area GABA neurons.

GABAergic neurons in mouse ventral tegmental area (VTA) exhibit elevated activity during withdrawal following chronic ethanol exposure. While increased glutamatergic input and decreased GABAA receptor sensitivity have been implicated, the impact of inhibitory signaling in VTA GABA neurons has not been fully addressed. We used electrophysiological and ultrastructural approaches to assess the impact of chronic intermittent ethanol vapour exposure in mice on GABAergic transmission in VTA GABA neurons during withdrawal. We used CRISPR/Cas9 ablation to mimic a somatodendritic adaptation involving the GABAB receptor (GABABR) in ethanol-naïve mice to investigate its impact on anxiety-related behaviour. The frequency of spontaneous inhibitory postsynaptic currents was reduced in VTA GABA neurons following chronic ethanol treatment and this was reversed by GABABR inhibition, suggesting chronic ethanol strengthens the GABABR-dependent suppression of GABAergic input to VTA GABA neurons. Similarly, paired-pulse depression of GABAA receptor-dependent responses evoked by optogenetic stimulation of nucleus accumbens inputs from ethanol-treated mice was reversed by GABABR inhibition. Somatodendritic currents evoked in VTA GABA neurons by GABABR activation were reduced following ethanol exposure, attributable to the suppression of GIRK (Kir3) channel activity. Mimicking this adaptation enhanced anxiety-related behaviour in ethanol-naïve mice. Chronic ethanol weakens the GABAergic regulation of VTA GABA neurons in mice via pre- and postsynaptic mechanisms, likely contributing to their elevated activity during withdrawal and expression of anxiety-related behaviour. As anxiety can promote relapse during abstinence, interventions targeting VTA GABA neuron excitability could represent new therapeutic strategies for treatment of alcohol use disorder.

The Kir channel in the nucleus tractus solitarius integrates the chemosensory system with REM sleep executive machinery for homeostatic balance

The locus coeruleus (LC), nucleus tractus solitarius (NTS), and retrotrapezoid nucleus (RTN) are critical chemosensory regions in the brainstem. In the LC, acid-sensing ion channels and proton pumps serve as H+ sensors and facilitate the transition from non-rapid eye movement (NREM) to rapid eye movement (REM) sleep. Interestingly, the potassium inward rectifier (KIR) channels in the LC, NTS, and RTN also act as H+-sensors and are a primary target for improving sleep in obstructive sleep apnea and Rett syndrome patients. However, the role of Kir channels in NREM to REM sleep transition for H+ homeostasis is not known. Male Wistar rats were surgically prepared for chronic sleep–wake recording and drug delivery into the LC, NTS, and RTN. In different animal cohorts, microinjections of the Kir channel inhibitor, barium chloride (BaCl2), at concentrations of 1 mM (low dose) and 2 mM (high dose) in the LC and RTN significantly increased wakefulness and decreased NREM sleep. However, BaCl2 microinjection into the LC notably reduced REM sleep, whereas it didn’t change in the RTN-injected group. Interestingly, BaCl2 microinjections into the NTS significantly decreased wakefulness and increased the percent amount of NREM and REM sleep. Additionally, with the infusion of BaCl2 into the NTS, the mean REM sleep episode numbers significantly increased, but the length of the REM sleep episode didn’t change. These findings suggest that the Kir channels in the NTS, but not in the LC and RTN, modulate state transition from NREM to REM sleep.

The vasodilator effect of Eugenol on uterine artery – potential therapeutic applications in pregnancy-associated hypertension

Preeclampsia, a gestational associated hypertension, has been reported in 6–8% of pregnant women worldwide leading to premature delivery and low birth weight of newborn due to reduced blood flow to placenta. Although several vasodilators (Methyl dopa, hydralazine, β-blockers and diuretics) are currently in use to treat preeclampsia, still there is a search for safer drugs with better efficacy. Lately, antihypertensive vasodilators from natural sources are gaining importance in treating preeclampsia. Eugenol (Eug), a natural essential oil, has been traditionally used in health and food products without any risk. In the present study, ex vivo experiments were designed to examine the vasorelaxation effect of Eug and its signaling pathways in a middle uterine artery (MUA) of pregnant Capra hircus (Ch). In presence of different blockers (L-NAME, indomethacin, ODQ, Ouabain, glibenclamide, 4-AP, Ba2, Carbenoxolone and 18β Glycyrrhetinic acid), Eug-induced concentration-dependent vasorelaxation response was elicited. The results showed that Eug caused a greater vasorelaxation effect in the MU of pregnant animals, which is mediated by potential activation of eNOS, KATP channels, and Kir channels with moderate activation of Na+- K+- ATPase and sGC and MEGJ. These findings provide a strong basis for developing Eug as a therapeutic candidate in the treatment of pregnancy-associated hypertension.

Endocannabinoid regulation of inward rectifier potassium (Kir) channels.

The inward rectifier potassium channel Kir2.1 (KCNJ2) is an important regulator of resting membrane potential in both excitable and non-excitable cells. The functions of Kir2.1 channels are dependent on their lipid environment, including the availability of PI(4,5)P2, secondary anionic lipids, cholesterol and long-chain fatty acids acyl coenzyme A (LC-CoA). Endocannabinoids are a class of lipids that are naturally expressed in a variety of cells, including cardiac, neuronal, and immune cells. While these lipids are identified as ligands for cannabinoid receptors there is a growing body of evidence that they can directly regulate the function of numerous ion channels independently of CBRs. Here we examine the effects of a panel of endocannabinoids on Kir2.1 function and demonstrate that a subset of endocannabinoids can alter Kir2.1 conductance to varying degrees independently of CBRs. Using computational and Surface plasmon resonance analysis, endocannabinoid regulation of Kir2.1 channels appears to be the result of altered membrane properties, rather than through direct protein-lipid interactions. Furthermore, differences in endocannabinoid effects on Kir4.1 and Kir7.1 channels, indicating that endocannabinoid regulation is not conserved among Kir family members. These findings may have broader implications on the function of cardiac, neuronal and/or immune cells.

Vasorelaxant mechanisms of the antidiabetic anagliptin in rabbit aorta: roles of Kv channels and SERCA pump.

The present study investigated the vasorelaxant mechanisms of an oral antidiabetic drug, anagliptin, using phenylephrine (Phe)-induced pre-contracted rabbit aortic rings. Arterial tone measurement was performed in rabbit thoracic aortic rings. Anagliptin induced vasorelaxation in a dose-dependent manner. Pre-treatment with the classical voltagedependent K+ (Kv) channel inhibitors 4-aminopyridine and tetraethylammonium significantly decreased the vasorelaxant effect of anagliptin, whereas pre-treatment with the inwardly rectifying K+ (Kir) channel inhibitor Ba2+, the ATP-sensitive K+ (KATP) channel inhibitor glibenclamide, and the large-conductance Ca2+-activated K+ (BKCa) channel inhibitor paxilline did not attenuate the vasorelaxant effect. Furthermore, the vasorelaxant response of anagliptin was effectively inhibited by pre-treatment with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid. Neither cAMP/protein kinase A (PKA)-related signaling pathway inhibitors (adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor KT 5720) nor cGMP/protein kinase G (PKG)-related signaling pathway inhibitors (guanylyl cyclase inhibitor ODQ and PKG inhibitor KT 5823) reduced the vasorelaxant effect of anagliptin. Similarly, the anagliptin-induced vasorelaxation was independent of the endothelium. Based on these results, we suggest that anagliptin-induced vasorelaxation in rabbit aortic smooth muscle occurs by activating Kv channels and the SERCA pump, independent of other vascular K+ channels, cAMP/PKA- or cGMP/PKG-related signaling pathways, and the endothelium.

Ionic mechanisms involved in arginine vasopressin-mediated excitation of auditory cortical and thalamic neurons

The axons containing arginine vasopressin (AVP) from the hypothalamus innervate a variety of structures including the cerebral cortex, thalamus, hippocampus and amygdala. A plethora amount of evidence indicates that activation of the V1a subtype of the vasopressin receptors facilitates anxiety-like and fear responses. As an essential structure involved in fear and anxiety responses, the amygdala, especially the lateral nucleus of amygdala (LA), receives glutamatergic innervations from the auditory cortex and auditory thalamus where high density of V1a receptors have been detected. However, the roles and mechanisms of AVP in these two important areas have not been determined, which prevents the understanding of the mechanisms whereby V1a activation augments anxiety and fear responses. Here, we used coronal brain slices and studied the effects of AVP on neuronal activities of the auditory cortical and thalamic neurons. Our results indicate that activation of V1a receptors excited both auditory cortical and thalamic neurons. In the auditory cortical neurons, AVP increased neuronal excitability by depressing multiple subtypes of inwardly rectifying K+ (Kir) channels including the Kir2 subfamily, the ATP-sensitive K+ channels and the G protein-gated inwardly rectifying K+ (GIRK) channels, whereas activation of V1a receptors excited the auditory thalamic neurons by depressing the Kir2 subfamily of the Kir channels as well as activating the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and a persistent Na+ channel. Our results may help explain the roles of V1a receptors in facilitating fear and anxiety responses.Categories: Cell Physiology.

Angiotensin II-Type-1a Receptor and Renal K + Wasting during Overnight Low-Na + Intake.

Chronic Angiotensin-II (Ang-II) perfusion stimulates Kir4.1/Kir5.1 of the DCT via angiotensin-II-type-1a-receptor (AT1aR) and low-sodium-intake also stimulates Kir4.1/Kir5.1. However, it is not explored the role of AT1aR in mediating the effect of LS on Kir4.1/Kir5.1. We used patch-clamp-technique to examine Kir4.1/Kir5.1 activity of the DCT, employed immunoblotting to examine NCC expression/activity, and used in vivo perfusion-technique to measure renal-Na+ and renal-K+-excretion in control, kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) and DCT-specific-AT1aR-knockout mice (DCT-AT1aR- KO). Ang-II acutely stimulated 40-pS-K+ channel (Kir4.1/Kir5.1-heterotetramer), increased whole-cell Kir4.1/Kir5.1-mediated K+-currents and the negativity of DCT-membrane-potential only in late-DCT2 but not in early-DCT. Acute Ang-II increased thiazide-induced renal Na+-excretion (ENa). The effect of Ang-II on Kir4.1/Kir5.1 and HCTZ-induced-ENa was absent in Ks-AT1aR-KO mice. Overnight-low-salt stimulated the expression of Agtr1a mRNA in DCT, increased whole-cell Kir4.1/Kir5.1-mediated K+-currents in late-DCT, hyperpolarized late-DCT membrane, augmented the expression of phosphor-Na-Cl-cotransporter (pNCC) and enhanced thiazide-induced renal-ENa in the control mice. However, the effect of overnight-low-salt on Kir4.1/Kir5.1-activity, DCT membrane potential and NCC activity/expression was abolished in DCT-AT1aR-KO or Ks-AT1aR-KO mice. Overnight-low-salt had no effect on baseline renal K+-excretion (EK) and plasma K+-concentrations in the control mice but it increased baseline renal-EK and decreased plasma K+-concentrations in DCT-AT1aR-KO or in Ks-AT1aR-KO mice. Acute Ang-II or overnight-LS stimulated Kir4.1/Kir5.1 in late-DCT and that AT1aR was responsible for acute Ang-II or overnight-low-salt-induced stimulation of Kir4.1/Kir5.1 and NCC. AT1aR of the DCT plays a role in maintaining adequate baseline renal-EK and plasma K+ concentrations during overnight-LS.

Structure of the Ion Channel Kir7.1 and Implications for its Function in Normal and Pathophysiologic States.

Hereditary defects in the function of the Kir7.1 in the retinal pigment epithelium are associated with the ocular diseases retinitis pigmentosa, Leber congenital amaurosis, and snowflake vitreal degeneration. Studies also suggest that Kir7.1 may be regulated by a GPCR, the melanocortin-4 receptor, in certain hypothalamic neurons. We present the first structures of human Kir7.1 and describe the conformational bias displayed by two pathogenic mutations, R162Q and E276A, to provide an explanation for the basis of disease and illuminate the gating pathway. We also demonstrate the structural basis for the blockade of the channel by a small molecule ML418 and demonstrate that channel blockade in vivo activates MC4R neurons in the paraventricular nucleus of the hypothalamus (PVH), inhibiting food intake and inducing weight loss. Preliminary purification, and structural and pharmacological characterization of an in tandem construct of MC4R and Kir7.1 suggests that the fusion protein forms a homotetrameric channel that retains regulation by liganded MC4R molecules.

Direct modulation of G protein-gated inwardly rectifying potassium (GIRK) channels.

Ion channels play a pivotal role in regulating cellular excitability and signal transduction processes. Among the various ion channels, G-protein-coupled inwardly rectifying potassium (GIRK) channels serve as key mediators of neurotransmission and cellular responses to extracellular signals. GIRK channels are members of the larger family of inwardly-rectifying potassium (Kir) channels. Typically, GIRK channels are activated via the direct binding of G-protein βγ subunits upon the activation of G-protein-coupled receptors (GPCRs). GIRK channel activation requires the presence of the lipid signaling molecule, phosphatidylinositol 4,5-bisphosphate (PIP2). GIRK channels are also modulated by endogenous proteins and other molecules, including RGS proteins, cholesterol, and SNX27 as well as exogenous compounds, such as alcohol. In the last decade or so, several groups have developed novel drugs and small molecules, such as ML297, GAT1508 and GiGA1, that activate GIRK channels in a G-protein independent manner. Here, we aim to provide a comprehensive overview focusing on the direct modulation of GIRK channels by G-proteins, PIP2, cholesterol, and novel modulatory compounds. These studies offer valuable insights into the underlying molecular mechanisms of channel function, and have potential implications for both basic research and therapeutic development.

MTORc2 in Distal Convoluted Tubule and Renal K + Excretion during High Dietary K + Intake.

Renal mTORc2 plays a role in regulating renal K+-excretion (renal-EK) and K+-homeostasis. Inhibition of renal mTORc2 caused hyperkalemia due to suppressing epithelial-Na+-channel (ENaC) and ROMK (Kir1.1) in the collecting duct. We now explore whether mTORc2 of DCT regulates basolateral Kir4.1/Kir5.1, NCC and renal-EK. We used patch-clamp-technique to examine basolateral Kir4.1/Kir5.1 in early-DCT, immunoblotting and immunofluorescence to examine NCC expression and in vivo measurement of urinary K+-excretion to determine baseline renal-EK in the mice treated with mTORc2-inhibitor and in DCT-specific Rapamycin-Insensitive-Companion- of-mTOR knockout (DCT-RICTOR-KO) mice. Inhibition of mTORc2 with AZD8055 abolished high-K+-induced inhibition of Kir4.1/Kir5.1 in DCT, high-K+-induced depolarization of DCT membrane and high-K+-induced suppression of pNCC expression. AZD8055 stimulated the 40-pS-inwardly-rectifying-K+ channel (Kir4.1/Kir5.1-heterotetramer) in early-DCT in the mice on overnight-high-K+, this effect was absent in the presence of PKC-inhibitor which also stimulated Kir4.1/Kir5.1. AZD8055-treatment decreased renal-EK in animals on overnight-high-K+. Deletion of RICTOR in the DCT increased the Kir4.1/Kir5.1-mediated K+-currents, hyperpolarized DCT membrane and increased the expression of pWNK4 and pNCC. Renal-EK was lower and plasma-K+ was higher in DCT-RICTOR-KO mice than corresponding control mice. Also, overnight-high-K+ did not inhibit Kir4.1/Kir5.1 activity in the DCT and failed to inhibit the expression of pNCC in DCT-RICTOR-KO mice. Overnight-high-K+ stimulated renal-EK in control mice, but this effect was attenuated in DCT-RICTOR-KO mice. Thus, overnight-high-K+ induced hyperkalemia in DCT-RICTOR-KO mice but not in control mice. mTORc2 of the DCT inhibits Kir4.1/Kir5.1 activity and NCC expression, and stimulates renal-EK during high-K+-intake.

The Impact of Chronic Magnesium Deficiency on Excitable Tissues-Translational Aspects.

Neuromuscular excitability is a vital body function, and Mg2+ is an essential regulatory cation for the function of excitable membranes. Loss of Mg2+ homeostasis disturbs fluxes of other cations across cell membranes, leading to pathophysiological electrogenesis, which can eventually cause vital threat to the patient. Chronic subclinical Mg2+ deficiency is an increasingly prevalent condition in the general population. It is associated with an elevated risk of cardiovascular, respiratory and neurological conditions and an increased mortality. Magnesium favours bronchodilation (by antagonizing Ca2+ channels on airway smooth muscle and inhibiting the release of endogenous bronchoconstrictors). Magnesium exerts antihypertensive effects by reducing peripheral vascular resistance (increasing endothelial NO and PgI2 release and inhibiting Ca2+ influx into vascular smooth muscle). Magnesium deficiency disturbs heart impulse generation and propagation by prolonging cell depolarization (due to Na+/K+ pump and Kir channel dysfunction) and dysregulating cardiac gap junctions, causing arrhythmias, while prolonged diastolic Ca2+ release (through leaky RyRs) disturbs cardiac excitation-contraction coupling, compromising diastolic relaxation and systolic contraction. In the brain, Mg2+ regulates the function of ion channels and neurotransmitters (blocks voltage-gated Ca2+ channel-mediated transmitter release, antagonizes NMDARs, activates GABAARs, suppresses nAChR ion current and modulates gap junction channels) and blocks ACh release at neuromuscular junctions. Magnesium exerts multiple therapeutic neuroactive effects (antiepileptic, antimigraine, analgesic, neuroprotective, antidepressant, anxiolytic, etc.). This review focuses on the effects of Mg2+ on excitable tissues in health and disease. As a natural membrane stabilizer, Mg2+ opposes the development of many conditions of hyperexcitability. Its beneficial recompensation and supplementation help treat hyperexcitability and should therefore be considered wherever needed.

Potassium Ion Channel Modulation as a Potential Remedy for Cadmium-induced Hypertension

Objective: Hypertension remains a chief non-communicable lifestyle disease that is negatively associated with dietary cadmium (Cd). The exact mechanism of Cd toxicity and the mitigating role of potassium supplementation are not fully known, hence, this study aimed to investigate their effects on the aorta. Hypothesis: We hypothesized that potassium supplementation can modulate Cd-induced vascular dysfunction. Method: Six cohorts of control, Cd-exposed, and potassium supplemented male Sprague-Dawley rats were selected. Hypertension was induced using cadmium chloride (2.5 or 5 mg/kg b.w.). Blood pressure was measured twice weekly. Reactivity to pharmacological agents with and without potassium ion channel modulation was assessed in thoracic aortic rings after eight weeks, using an organ bath set-up. Data: Statistical analysis was done using one-way analysis of variance and the results were reported as mean ± standard error of the mean. The Games-Howell or Bonferroni post hoc test was used for multiple comparisons. Summary of Results: Cadmium significantly (p<0.05) elevated systolic, diastolic, mean arterial, and pulse pressures, reduced (p<0.001) phenylephrine-induced contraction mostly via BKCa channels, and augmented (p<0.05) sodium nitroprusside-induced relaxation via K-ATP, KV, and KIR channels, without affecting acetylcholine-induced relaxation. Potassium supplementation ameliorated these effects via KIR and BKCa channels. Conclusions: The results indicate that modulation of potassium ion channels in the vasculature might provide therapeutic management for environmentally-induced hypertension and should be further explored. Funding source: The Mona Campus Committee for Research and Publications and Graduate Awards, School for Graduate Studies and Research, University of the West Indies. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gene Expression, Morphology, and Electrophysiology During the Dynamic Development of Human Induced Pluripotent Stem Cell-Derived Atrial- and Ventricular-Like Cardiomyocytes.

Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived atrial- and ventricular-like cardiomyocytes (iPS-AM and iPS-VM, respectively). For iPS-AM/VM differentiation, we performed the small molecule-based temporal modulation of the retinoic acid and bone morphogenetic protein signaling pathways. We investigated the gene expression and morphology using immunofluorescence, quantitative real-time polymerase chain reaction, flow cytometry, and transmission electron microscopy as well as registered electrophysiological functions using a whole-cell patch clamp on days 20, 30, and 60 post-differentiations. Pan-cardiomyocyte marker, including troponin T2 (TNNT2) and alpha-actinin-2 (ACTN2), expressions increased both in iPS-AMs and iPS-VMs. Similarly, the mRNA expression of both iPS-AM-specific markers, ie, natriuretic peptide A (NPPA), myosin light chain 7 (MYL7), and K+ channel Kir3.4 (KCNJ5), and iPS-VM-specific markers, ie, gap junction α-1 (GJA1), myosin light chain 2 (MYL2), and alpha-1-subunit of a voltage-dependent L-type calcium channel (CACNA1C), increased from 0 to 20 days, and then decreased from 30 to 60 days. Concerning morphology, cardiac troponin-T (cTnT) arrangement was progressively organized and developed from a disorderly myofibrillar distribution to an organized sarcomere pattern both in iPS-AMs and iPS-VMs. Mitochondrial numbers gradually increased and those of lipid droplets decreased during dynamic development. Regarding physiological function, the resting and action potential amplitudes remained statistically indifferent in both cell types, and the action potential duration was prolonged during the development. IPS-AMs/VMs displayed dynamic development concerning their gene expression, morphology, and electrophysiological function. The discoveries of this study could provide novel insights into heart development and encourage further research.

Effects of potassium supplementation and deficiency on the progression of salt-sensitive hypertension

While the physiological effects of sodium on blood pressure are well established, many of the effects of potassium remain unclear. Prior work found that increased potassium intake decreases blood pressure and risk of cardiovascular disease, improves vasodilation, has reno-protective effects, and can stimulate natriuresis. In this study, we examined how different sodium/potassium ratios affect the development of salt-sensitive hypertension, specifically examining changes in the kidney and the renin angiotensin aldosterone system (RAAS). We hypothesized that blood pressure would directly correlate to the Na+/K+ ratio and that there would be compensatory changes in RAAS and in expression of renal potassium channels, particularly inwardly rectifying potassium (Kir) channels. At 9 weeks of age, male and female Dahl SS rats were placed on a high salt (4% NaCl) diet for 5 weeks with normal potassium (NK, 0.36% K+), high potassium (HK, 1.41% K+), or potassium depleted (DK, <0.0001% K+) diet. After 5 weeks of dietary intervention, DK animals were hypokalemic, and HK animals had significantly increased plasma potassium. Potassium supplementation attenuated the development of salt-sensitive hypertension in male rats fed the HK diet compared to NK; the DK group did not develop hypertension. Potassium supplementation did not attenuate blood pressure in female rats, who also had a higher mortality rate on the DK diet. Heart rates were unchanged in both male and female HK groups but were significantly lower in the DK groups of both sexes. After 5 weeks of diet, the total body weights of DK rats were significantly lower. The 2 kidney/total body weight ratios were not different between the HK and NK groups, but the kidneys of the DK group were significantly larger, likely due to their smaller body weights. Medullary protein casts in DK rats of both sexes were decreased but no renoprotective effects were observed in the HK group. Western blot analysis of renal potassium channels demonstrated no changes in the expression of ROMK, significantly increased Kir5.1 in DK males and females, and a significant increase in Kir4.1 and the alpha-subunit of the Na+/K+-ATPase in DK males. We analyzed RAAS metabolites from plasma samples collected at the endpoint: Ang I (1-10), Ang IV (3-8), and Ang (1-5) were significantly decreased in DK males and females, Ang II (1-8) and Ang III (2-8) were significantly reduced in DK males, and aldosterone was significantly increased in HK males and females. These results provide a comprehensive assessment of how various potassium diets affect the development of salt-sensitive hypertension in both males and females, which will provide a solid foundation for future mechanistic studies. R01DK129227, I01 BX004024, R35HL135749. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Restoring capillary-to-arteriole signaling in Alzheimer's Disease

There is growing evidence for vascular contributions to cognitive impairments in Alzheimer’s Disease (AD). Functional hyperemia, which refers to local increases in blood flow in response to local increases in brain activity, is impaired early in patients and animal models of AD. The mechanisms underlying functional hyperemia are referred to as neurovascular coupling (NVC). A key player in NVC is the inward rectifying K+ channel (Kir2.1) in capillary endothelial cells (cECs). Activation of Kir2.1 channels initiates a retrograde hyperpolarizing signal which causes upstream arterioles to dilate and increase local blood flow. Studies have shown that Kir2.1 channel function is impaired in the 5xFAD mouse model for AD, and administration of Kir2.1 cofactor, phosphatidylinositol 4,5-bisphosphate (PIP2), was suffcient to restore channel function and functional hyperemia deficits. The objective of this study is to characterize the effect of AD on NVC. Since AD is characterized by the deposition of amyloid beta (Aβ), we hypothesize that Aβ is impairing Kir2.1 function in cECs at an early stage of the disease. Using 6-month-old female and male 5xFAD mice, we performed our ex vivo capillary-parenchymal arteriole (CaPA) technique to investigate NVC responses by directly stimulating capillaries and recording upstream arteriolar dilation in real time. We further tested the direct effect of Aβ oligomers (200 nM) and reactive oxygen species (ROS) scavengers (superoxide dismutase 120 U/mL + catalase 1000 U/mL), to probe their effect on the NVC response. Finally, to test the ability of PIP2 to rescue the NVC response, systemic administration via intraperitoneal injection was performed one hour before CaPA experiment. Consistent with a NVC impairment in AD, capillary stimulation with 10 mM K+ failed to evoke upstream arteriolar dilation in 5xFAD mice at an early stage, and this impairment was recapitulated in wild type mice after treatment with Aβ. Importantly, Aβ only caused a modest arteriole constriction (20.11% ± 5.24 %) but did not impair its ability to dilate in response to direct stimulation with 10 mM K+, suggesting an effect on cECs. ROS scavenging during Aβ treatment prevented NVC impairment as 10 mM K+ capillary stimulation induced an upstream arteriolar dilation of 59% of max dilation. Finally, systemic administration of PIP2 also rescued the NVC response to the levels observed in control conditions. Taken together, our results suggest that Aβ is causing impairment in NVC via increasing ROS production, specifically at the capillary endothelial level. This increase in ROS is likely leading to PIP2 degradation and causing Kir2.1 channel dysfunction. Administration of PIP2 was suffcient to rescue the NVC response, suggesting a possible strategy for rescuing functional hyperemia in AD. This study was supported by a research grant from the Center for Women’s Health Research located at the University of Colorado Anschutz Medical Campus; 2 research grants from the University of Pennsylvania Orphan Disease Center in partnership with the cureCADASIL (2019 and 2022), the National Institute of Neurological Disorders and Stroke RF1/R01NS129022, and the National Heart, Lung, and Blood Institute R01HL136636 to FD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MiRNA Regulation of Cerebrovascular Inwardly Rectifying K+ Channels During Onset of Alzheimer Disease: Role of Cholesterol and Cannabidiol Treatment

Currently, more than 6 million Americans live with Alzheimer disease (AD). Recent studies demonstrate that impaired blood flow in the brain is integral to the development of AD. Vascular inwardly rectifying K+ channels, such as KIR2.x and KIR6.x, are important for regulating vasoconstriction and vasodilation to tune cerebral perfusion in response to metabolic demand. We’ve previously identified that disruption in membrane cholesterol during advanced AD pathology restores function of KIR2.1 channels to that of young, healthy conditions or better. Additionally, we’ve identified specific microRNAs (miRNAs) in cerebral vessels that can be used to track early development of AD. In this study, we seek to understand molecular pathways stemming from miRNAs that directly or indirectly modulate activity of KIR2.x ( Kcnj2, Kcnj12) and KIR6.x ( Kcnj8, Kcnj11) channels. These ion channels are abundant in cerebrovascular endothelium and smooth muscle cells while particularly sensitive to dyslipidemia. We hypothesize that cerebrovascular miRNAs marking AD pathology are involved in dysregulation of inwardly rectifying K+ channels via cholesterol signaling. With employing QIAGEN’s Ingenuity Pathway Analysis (IPA), we first identified miRNAs that generally regulate each of the target genes for select KIR channels. With applying acquired knowledge of cerebrovascular miRNAs implicated in AD, we found which of these miRNAs also regulate cellular membrane cholesterol levels. Finally, we pinpointed miRNAs sensitive to cannabidiol (CBD) treatment during the development of AD as a potential therapeutic alternative to statins and cyclodextrins. In total, there are 233 miRNAs that regulate Kcnj2, 43 for Kcnj8, 203 for Kcnj11, and 177 for Kcnj12. When factoring in miRNAs that are AD-related, remaining miRNAs include nine that regulate Kcnj2, and one each for Kcnj8, Kcnj11, and Kcnj12. Remaining miRNAs that are cholesterol-related include one for Kcnj2, 43 for Kcnj8, 191 for Kcnj11, and one for Kcnj12. In general, at AD onset, CBD upregulates KIR2.2 and downregulates KIR2.1, KIR6.1, and KIR6.2 channels. CBD reversed expression of miRNAs involved in KIR2.x channel regulation in endothelial and smooth muscle/pericytes (e.g., miR-133 and miR-145) during AD onset in 3xTg-AD animals relative to wild-type controls. CBD also reverses expression of miR-129 and miR-151 to effectively upregulate genes modulating KIR6.x channels from onset of AD relative to wild-type. Altogether, we evidence specific pathways underpinning regulation of KIR2.x and KIR6.x channels in the transition from a healthy to diseased brain. This work was supported by the National Institutes of Health (R01AG073230). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Astroglial Kir4.1 potassium channel deficit drives neuronal hyperexcitability and behavioral defects in Fragile X syndrome mouse model

Fragile X syndrome (FXS) is an inherited form of intellectual disability caused by the loss of the mRNA-binding fragile X mental retardation protein (FMRP). FXS is characterized by neuronal hyperexcitability and behavioral defects, however the mechanisms underlying these critical dysfunctions remain unclear. Here, using male Fmr1 knockout mouse model of FXS, we identify abnormal extracellular potassium homeostasis, along with impaired potassium channel Kir4.1 expression and function in astrocytes. Further, we reveal that Kir4.1 mRNA is a binding target of FMRP. Finally, we show that the deficit in astroglial Kir4.1 underlies neuronal hyperexcitability and several behavioral defects in Fmr1 knockout mice. Viral delivery of Kir4.1 channels specifically to hippocampal astrocytes from Fmr1 knockout mice indeed rescues normal astrocyte potassium uptake, neuronal excitability, and cognitive and social performance. Our findings uncover an important role for astrocyte dysfunction in the pathophysiology of FXS, and identify Kir4.1 channel as a potential therapeutic target for FXS.

The Vasodilator Effect and Mechanism of 18β-glycyrrhetinic Acid on Isolated Pulmonary Artery Rings in Rats

Background: The purpose of this study was to evaluate the effect of 18β-glycyrrhetinic acid (18β-GA) on isolated rat pulmonary artery vascular rings as well as the mechanism that lies behind the diastolic effects of this compound. Methods: To examine the effects of various doses of 18β-GA on resting normal vascular rings, we used isolated rat pulmonary artery vascular rings. The isolated rat pulmonary artery vascular ring was precontracted with phenylephrine (PE) and potassium chloride solution (KCl), which allowed for the observation of the diastolic effects of 18β-GA, as well as the effects of various concentrations of 18β-GA on blocking the four potassium channels of glibenclamide, barium chloride (BaCl2), tetraethylamine (TEA), or 4-aminopyridine; the impact of various 18β-GA concentrations on the pulmonary artery vasodilation effect pre-use of the nitric oxide synthase inhibitors l-nitroarginine methyl ester and indomethacin. Results: It was shown that 18β-GA has concentration-dependent diastolic effects on isolated rat pulmonary vascular rings that have been precontracted with PE and KCl but it has no effect on resting isolated thoracic aortic vascular rings. Conclusion: The two Kir and Kv channels may be connected to the endothelium-dependent vasodilation mechanism of 18β-GA.

Dual pattern of cholesterol-induced decoupling of residue-residue interactions of Kir2.2

Cholesterol is a negative regulator of a variety of ion channels. We have previously shown that cholesterol suppresses Kir2.2 channels via residue-residue uncoupling on the inter-subunit interfaces within the close state of the channels (3JYC). In this study, we extend this analysis to the other known structure of Kir2.2 that is closer to the open state of Kir2.2 channels (3SPI) and provide additional analysis of the residue distances between the uncoupled residues and cholesterol binding domains in the two conformation states of the channels. We found that the general phenomenon of cholesterol binding leading to uncoupling between specific residues is conserved in both channel states but the specific pattern of the uncoupling residues is distinct between the two states and implies different mechanisms. Specifically, we found that cholesterol binding in the 3SPI state results in an uncoupling of residues in three distinct regions; the transmembrane domain, membrane-cytosolic interface, and the cytosolic domain, with the first two regions forming an envelope around PI(4,5)P2 and cholesterol binding sites and the distal region overlapping with the subunit-subunit interface characterized in our previous study of the disengaged state. We also found that this uncoupling is dependent upon the number of cholesterol molecules bound to the channel. We further generated a mutant channel Kir2.2P187V with a single point mutation in a residue proximal to the PI(4,5)P2 binding site, which is predicted to be uncoupled from other residues in its vicinity upon cholesterol binding and found that this mutation abrogates the sensitivity of Kir2.2 to cholesterol changes in the membrane. These findings suggest that cholesterol binding to this conformation state of Kir2.2 channels may destabilize the PI(4,5)P2 interactions with the channels while in the disengaged state the destabilization occurs where the subunits interact. These findings give insight into the structural mechanistic basis for the functional effects of cholesterol binding to the Kir2.2 channel.