Kir Channel Function Research Articles

Mechanisms underlying the distinct K+ dependencies of periodic paralysis.

Patients with periodic paralysis have attacks of weakness precipitated by depolarization of muscle. Each form of periodic paralysis is associated with unique changes in serum K+ during attacks of weakness. In hypokalemic periodic paralysis (hypoKPP), the mutation-induced gating pore current causes weakness associated with low serum K+. In hyperkalemic periodic paralysis (hyperKPP), mutations increase a non-inactivating Na+ current (Na persistent or NaP), which causes weakness associated with elevation of extracellular K+. In Andersen-Tawil syndrome, mutations causing loss of Kir channel function cause weakness associated with either low or high K+. We developed a computer model to address two questions: (1) What mechanisms are responsible for the distinct K+ dependencies of muscle depolarization-induced weakness in the three forms of periodic paralysis? (2) Why does extracellular K+ become elevated during attacks of weakness in hyperKPP, reduced in hypoKPP, and both elevated and reduced in Andersen-Tawil syndrome? We experimentally tested the model assumptions about resting potential in normal K+ solution in hyperKPP and hypoKPP. Recreating the distinct K+ dependence of all three forms of periodic paralysis required including the K+ and voltage dependence of current through Kir channels, the extracellular K+ and intracellular Na+ dependence of the Na/K ATPase activity, and the distinct voltage dependencies of the gating pore current and NaP. A key factor determining whether muscle would depolarize was the direction of small net K+ and net Na+ fluxes, which altered ion concentrations over hours. Our findings may aid in development of novel therapy for diseases with dysregulation of muscle excitability.

Preeclamptic serum and soluble fms-like tyrosine kinase-1 suppress endothelial inward rectifier potassium currents

IntroductionInward rectifier K+ (Kir) channel, a major factor determining endothelial membrane potential, regulates Ca2+ influx and vasodilator release, which is impaired in preeclamptic blood vessels. Previously, human umbilical vein endothelial cell (HUVEC) Kir currents were shown to decrease after incubating in preeclamptic plasma. We aimed to demonstrate whether sFlt-1, which is high in preeclamptic blood, could inhibit Kir channel function and expression. MethodsHUVECs were cultured in regular medium, regular medium with added sFlt-1, or serum from preeclampsia patients or normal pregnant women (Control, sFlt-1, PE, or NP, respectively). Using whole-cell patch clamp technique, we identified Kir currents with the Kir blocker 2 mM BaCl2 and compared the currents among groups. The expression of Kir 2.1 and 2.2 channels were determined using immunofluorescent staining. ResultssFlt-1 and PE groups exhibited similar Kir currents, while NP group possessed significantly larger currents, similar to Control group currents. Moreover, sFlt-1 and sFlt-1/PlGF ratio showed strong negative correlation with Kir currents (r = −0.71 and −0.70, respectively; P < 0.05). There were no significant differences in mean fluorescence intensity representing Kir 2.1 and 2.2 channels expression in all four groups. DiscussionThis is the first report to demonstrate sFlt-1 inhibition against Kir currents, which could lead to maternal endothelial dysfunction and hypertension seen in preeclampsia. However, channel expression was unaffected by sFlt-1 incubation, suggesting dysfunctions of channel or other processes (e.g., membrane translocation). The present data could pave the way for novel therapies targeting sFlt-1 or Kir to alleviate hypertension in preeclampsia.

Alzheimer's disease and cerebrovascular pathology alter inward rectifier potassium (KIR 2.1) channels in endothelium of mouse cerebral arteries.

Background and PurposeInward rectifier potassium (KIR) channels are key effectors of vasodilatation in neurovascular coupling (NVC). KIR channels expressed in cerebral endothelial cells (ECs) have been confirmed as essential modulators of NVC. Alzheimer's disease (AD) and cerebrovascular disease (CVD) impact on EC‐KIR channel function, but whether oxidative stress or inflammation explains this impairment remains elusive.Experimental ApproachWe evaluated KIR channel function in intact and EC‐denuded pial arteries of wild‐type (WT) and transgenic mice overexpressing a mutated form of the human amyloid precursor protein (APP mice, recapitulating amyloid β‐induced oxidative stress seen in AD) or a constitutively active form of TGF‐β1 (TGF mice, recapitulating inflammation seen in cerebrovascular pathology). The benefits of antioxidant (catalase) or anti‐inflammatory (indomethacin) drugs also were investigated. Vascular and neuronal components of NVC were assessed in vivo.Key ResultsOur findings show that (i) KIR channel‐mediated maximal vasodilatation in APP and TGF mice reaches only 37% and 10%, respectively, of the response seen in WT mice; (ii) KIR channel dysfunction results from KIR2.1 subunit impairment; (iii) about 50% of K+‐induced artery dilatation is mediated by EC‐KIR channels; (iv) oxidative stress and inflammation impair KIR channel function, which can be restored by antioxidant and anti‐inflammatory drugs; and (v) inflammation induces KIR2.1 overexpression and impairs NVC in TGF mice.Conclusion and ImplicationsTherapies targeting both oxidative stress and inflammation are necessary for full recovery of KIR2.1 channel function in cerebrovascular pathology caused by AD and CVD.

Methyl-Beta-Cyclodextrin Restores KIR Channel Function in Brain Endothelium of Female Alzheimer's Disease Mice.

Background:As the sixth-leading cause of death in the United States, Alzheimer’s disease (AD) entails deteriorating endothelial control of blood flow throughout the brain. In particular, reduced inward-rectifying K+ (KIR) channel function in animal models of aging and AD compromises endothelial function and optimal perfusion of brain parenchyma. Deficient endothelial KIR channels may result from aberrant interaction with plasma membrane cholesterol as a primary regulator of membrane fluidity and ion channels.Objective:We tested the hypothesis that mild methyl-β-cyclodextrin (MβCD) treatment to reduce membrane cholesterol may restore endothelial KIR channel function in brain endothelium of old AD mice.Methods:Membrane potential was continuously measured in isolated endothelial tubes from posterior cerebral arteries of young (1 to 3 months) and old (16 to 19 months) female 3xTg-AD mice before and after mild treatment with the cholesterol-removing agent MβCD (1 mmol/L). Elevated extracellular potassium ([K+]E; 15 mmol/L) and NS309 (1μmol/L) activated KIR and Ca2+-activated K+ (SKCa/IKCa) channels respectively before and after MβCD treatment.Results:SKCa/IKCa channel function for producing hyperpolarization remained stable regardless of age group and MβCD treatment (ΔVm: ∼–33 mV). However, as deficient during AD, KIR channel function was restored (ΔVm: –9±1 mV) versus pre-MβCD conditions (–5±1 mV); a progressive effect that reached –14±1 mV hyperpolarization at 60 min following MβCD washout.Conclusion:In female animals, MβCD treatment of brain endothelium selectively restores KIR versus SKCa/IKCa channel function during AD. Thus, the endothelial cholesterol-KIR channel interface is a novel target for ameliorating perfusion of the AD brain.

Activity of Inwardly Rectifying K + Channels in Cerebral Arteries is Diminished in Dyslipidemia Without Overt Effects on Cerebral Blood Flow

Endothelial dysfunction associated with dyslipidemia is a major contributor to cardiovascular disease. Dilatory function of resistance arteries is diminished in dyslipidemia through altered activity of ion channels that set membrane potential. We examined whether inwardly rectifying K+ (KIR) channels are targeted early in dyslipidemia and if alterations impact cerebral blood flow control. Experiments began at the cellular level (patch-clamp electrophysiology) and then were extended to isolated arteries (pressure myography) and whole animals (arterial spin-labelling magnetic resonance imaging). Initial lipid analysis confirmed the dyslipidemic state of LDLR-/- (normal chow) and C57BL/6 mice fed a high-cholesterol high-fat (HFHC) diet for 8 weeks; note aortic plaque formation was absent in these animal models. Patch-clamp electrophysiology revealed a marked reduction in endothelial but not smooth muscle KIR activity in both dyslipidemic models. Flow activation of KIR was also reduced in endothelial cells from LDLR-/- and HFHC mice. However, endothelial KIR activity was recovered in dyslipidemic mice by depleting the plasma membrane of cholesterol. Consistent with these cellular changes, we observed diminished shear-induced vasodilation in cerebral arteries isolated from dyslipidemic animals. However, these cellular and tissue level changes did not alter cerebral blood flow at baseline or in response to a blood pressure challenge (phenylephrine injection through a peritoneal catheter, 0.816 mg/kg). The maintenance of blood flow control was observed across a full range of structures including the cortex, cerebral nuclei, hippocampus, thalamus, hypothalamus and midbrain. Our findings highlight that while KIR channel function is targeted early on in dyslipidemia, compensatory mechanisms can maintain brain blood flow to preserve neurological function.

Removal of membrane cholesterol selectively restores K IR channel function in brain endothelium during Alzheimer's disease

The number of Alzheimer's disease (AD) patients (~44 million worldwide; ~5.8 million Americans) continues to rise, while there is no effective treatment available and etiology remains obscure. Incidence of AD increases with aging due, in large part, to deteriorating endothelial control of blood flow throughout the brain. Further, K+ channels, particularly inward-rectifying K+ (KIR2.x) channels, are integral to endothelial function and optimal perfusion of brain parenchyma. Our group has identified reduced KIR2.x channel function as a new manifestation of endothelial “dysfunction” during aging and AD, whereas function of small- and intermediate-Ca2+-activated K+ (SKCa/IKCa) channels is stable throughout. Moreover, we reasoned that apparently deficient KIR2.x channels could be due to their aberrant interaction with plasma membrane cholesterol, a major regulator of membrane fluidity and ion channels. Hence, we tested the hypothesis that reduction of membrane cholesterol may restore KIR channel function in brain endothelium of old AD mice. Membrane potential (Vm) was measured in posterior cerebral endothelial “tubes” of female 3xTg-AD mice (16 to 19 mo; n=12) before and after mild treatment with the cholesterol-removing agent methyl-β-cyclodextrin (MβCD; 1 mM). We used elevated extracellular potassium([K+]E;15 mM) and NS309 (1 µM) to activate KIR2.x and SKCa/IKCa channels respectively. SKCa/IKCa channel function for producing hyperpolarization (more negative Vm) remains stable after MβCD treatment (ΔVm, mV; -31±1 vs. control, -33±2), revealing that SKCa/IKCa channels are not significantly impacted by membrane cholesterol. However, KIR2.x channel function is progressively restored and enhanced in a time-dependent manner following washout of MβCD (ΔVm, mV; control, -5±1; 30 min washout, -9±1; 60 min, -14±1). Altogether, our findings demonstrate that cholesterol intervention of brain endothelium selectively restores KIR2.x vs. SKCa/IKCa channel function during AD. Thus, endothelial cholesterol-KIR channel interaction is a novel therapeutic target for ameliorating cerebrovascular function to optimize perfusion of the AD brain.

Phylogenomics of Tick Inward Rectifier Potassium Channels and Their Potential as Targets to Innovate Control Technologies.

This study was conducted to enhance the identification of novel targets to develop acaricides that can be used to advance integrated tick-borne disease management. Drivers for the emergence and re-emergence of tick-borne diseases affecting humans, livestock, and other domestic animals in many parts of the world include the increased abundance and expanded geographic distribution of tick species that vector pathogens. The evolution of resistance to acaricides among some of the most important tick vector species highlights the vulnerability of relying on chemical treatments for tick control to mitigate the health burden of tick-borne diseases. The involvement of inward rectifier potassium (Kir) channels in homeostasis, diuresis, and salivary gland secretion in ticks and other pests identified them as attractive targets to develop novel acaricides. However, few studies exist on the molecular characteristics of Kir channels in ticks. This bioinformatic analysis described Kir channels in 20 species of hard and soft ticks. Summarizing relevant investigations on Kir channel function in invertebrate pests allowed the phylogenomic study of this class of ion channels in ticks. How this information can be adapted to innovate tick control technologies is discussed.

Flonicamid and knockdown of inward rectifier potassium channel gene CsKir2B adversely affect the feeding and development of Chilo suppressalis.

The selective insecticide flonicamid shows highly insecticidal activities against piercing-sucking insects and has been widely used for the control of Hemipteran insect pests, whereas its effects on Lepidopteran insect pests remain largely unknown. Recently, inward rectifier potassium (Kir) channel has been verified to be a target of flonicamid, however, functional characterization of Lepidopteran Kir genes is still lacking. Flonicamid shows no insecticidal toxicity against Chilo suppressalis larvae. However, the feeding and growth of larvae were reversibly inhibited by flonicamid (50-1200 mg L-1 ). Flonicamid treatment also remarkably reduced and delayed the pupation and eclosion of Chilo suppressalis. Additionally, five distinct Kir channel genes (CsKir1, CsKir2A, CsKir2B, CsKir3A and CsKir3B) were cloned from Chilo suppressalis. Expression profiles analysis revealed that CsKir2A was predominately expressed in the hindgut of larvae, whereas CsKir2B had high expressions in the Malpighian tubules and hindgut. RNA interference (RNAi)-mediated knockdown of CsKir2B significantly reduced the growth and increased the mortalities of larvae, whereas silencing of CsKir2A had no obvious effects on Chilo suppressalis. Flonicamid exhibits adverse effects on the growth and development of Chilo suppressalis. CsKir2B might be involved in the feeding behavior of Chilo suppressalis. These results provide valuable information on the effects of flonicamid on non-target insects as well as the function of insect Kir channels, and are helpful in developing new insecticide targeting insect Kir channels. © 2020 Society of Chemical Industry.

Development of Alzheimer's Disease Progressively Alters Sex-Dependent KCa and Sex-Independent KIR Channel Function in Cerebrovascular Endothelium.

Development of Alzheimer's disease (AD) pathology is associated with impaired blood flow delivery of oxygen and nutrients throughout the brain. Cerebrovascular endothelium regulates vasoreactivity of blood vessel networks for optimal cerebral blood flow. We tested the hypothesis that cerebrovascular endothelial Gq-protein-coupled receptor (GPCR; purinergic and muscarinic) and K+ channel [Ca2+-activated (KCa2.3/SK3 and KCa3.1/IK1) and inward-rectifying (KIR2.x)] function declines during progressive AD pathology. We applied simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and membrane potential (Vm) in freshly isolated endothelium from posterior cerebral arteries of 3×Tg-AD mice [young, no pathology (1- 2 mo), cognitive impairment (CI; 4- 5 mo), extracellular Aβ plaques (Aβ; 6- 8 mo), and Aβ plaques + neurofibrillary tangles (AβT; 12- 15 mo)]. The coupling of ΔVm-to-Δ[Ca2+]i during AβT pathology was lowest for both sexes but, overall, ATP-induced purinergic receptor function was stable throughout AD pathology. SKCa/IKCa channel function itself was enhanced by ∼20% during AD (Aβ+ AβT) versus pre-AD (Young + CI) in males while steady in females. Accordingly, hyperpolarization-induced [Ca2+]i increases following SKCa/IKCa channel activation and Δ[Ca2+]i-to-ΔVm coupling was enhanced by ≥two-fold during AD pathology in males but not females. Further, KIR channel function decreased by ∼50% during AD conditions versus young regardless of sex. Finally, other than a ∼40% increase in females versus males during Aβ pathology, [Ca2+]i responses to the mitochondrial uncoupler FCCP were similar among AD versus pre-AD conditions. Altogether, AD pathology represents a condition of altered KCa and KIR channel function in cerebrovascular endothelium in a sex-dependent and sex-independent manner respectively.

Screening Technologies for Inward Rectifier Potassium Channels: Discovery of New Blockers and Activators.

K+ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In nonexcitable cells, K+ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K+ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K+ equilibrium potential (EK). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K+ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (KATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

From Cells‐to‐Organism: Impact of Dyslipidemia on Inwardly Rectifying K+ Channels and Cerebral Vascular Function

Recent studies have noted that dyslipidemia can diminish dilatory function of resistance arteries by altering the activity of ion channels that set membrane potential. Focusing on the cerebral circulation, this study determined whether inwardly rectifying K+ (KIR) channels are targeted early in dyslipidemia and if alterations impact blood flow control. Experiments began at the cellular level (patch‐clamp electrophysiology) and then were extended to isolated arteries (pressure myography) and whole animals (arterial spin‐labelling magnetic resonance imaging). Initial lipid analysis confirmed the dyslipidemic state of LDLR−/− (normal chow) and C57BL/6 mice fed a high‐cholesterol high‐fat (HFHC) diet for 8 weeks; note aortic plaque formation was absent in these animal models. Patch‐clamp electrophysiology revealed a marked reduction in endothelial but not smooth muscle KIR activity in both dyslipidemic models. Flow activation of KIR was also reduced in endothelial cells from LDLR−/− and HFHC mice. However, endothelial KIR activity was recovered in dyslipidemic mice by depleting the plasma membrane of cholesterol. Consistent with these cellular changes, we observed diminished shear‐induced vasodilation in cerebral arteries isolated from dyslipidemic animals. To our surprise, these cellular/tissue level changes didn’t alter cerebral blood flow at baseline or in response to a blood pressure challenge (phenylephrine injection through a peritoneal catheter, 0.816 mg/kg). The maintenance of blood flow control was observed across a full range of structures including the cortex, cerebral nuclei, hippocampus, thalamus, hypothalamus and midbrain. In closing, our findings highlight that while KIR channel function is targeted early on in dyslipidemia, compensatory mechanisms can maintain brain blood flow to preserve neurological function.Support or Funding InformationCanadian Institutes of Health Research

Aging Alters Cerebrovascular Endothelial GPCR and K+ Channel Function: Divergent Role of Biological Sex.

Age-related dementia entails impaired blood flow to and throughout the brain due, in part, to reduced endothelial nitric oxide signaling. However, it is unknown whether sex affects cerebrovascular Gq-protein-coupled receptors (GPCRs) and K+ channels underlying endothelium-derived hyperpolarization (EDH) during progressive aging. Thus, we simultaneously evaluated intracellular Ca2+ ([Ca2+]i) and membrane potential (Vm) of intact endothelial tubes freshly isolated from posterior cerebral arteries of young (4-6 mo), middle-aged (12-16 mo), and old (24-28 mo) male and female C57BL/6 mice. Purinergic receptor function (vs. muscarinic) was dominant and enhanced for [Ca2+]i increases in old females versus old males. However, Ca2+-sensitive K+ channel function as defined by NS309-evoked Vm hyperpolarization was mildly impaired in females versus males during old age. This sex-based contrast in declined function of GPCRs and K+ channels to produce EDH may support a greater ability for physiological endothelial GPCR function to maintain optimal cerebral blood flow in females versus males during old age. As reflective of the pattern of cerebral blood flow decline in human subjects, inward-rectifying K+ (KIR) channel function decreased with progressive age regardless of sex. Combined age-related analyses masked male versus female aging and, contrary to expectation, hydrogen peroxide played a minimal role. Altogether, we conclude a sex-based divergence in cerebrovascular endothelial GPCR and K+ channel function while highlighting a previously unidentified form of age-related endothelial dysfunction as reduced KIR channel function.

Chiral Specificity of Cholesterol Orientation Within Cholesterol Binding Sites in Inwardly Rectifying K+ Channels.

Cholesterol is an integral component of cellular membranes and has been shown to be an important functional regulator for many different ion channels, including inwardly rectifying potassium (Kir) channels. Consequently, understanding the molecular mechanisms underlying this regulation represents a critical field of study. Broadly speaking, cholesterol can mediate ion channel function either directly by binding to specific sites or indirectly by altering surrounding membrane properties. Owing to the similar effects of cholesterol and its chiral isomers (epicholesterol and ent-cholesterol) on membrane properties, comparative analysis of these sterols can be an effective tool for discriminating between these direct and indirect effects. Indeed, this strategy was used to demonstrate the direct effect of cholesterol on Kir channel function. However, while this approach can discriminate between direct and indirect effects, it does not account for the promiscuity of cholesterol binding sites, which can potentially accommodate cholesterol or its chiral isomers. In this chapter, we use docking analyses to explore the idea that the specificity of cholesterol's effect on Kir channels is dependent on the specific orientation of cholesterol within its putative binding pocket which its chiral isomers cannot replicate, even when bound themselves.

Increased Hydrogen Peroxide After Traumatic Brain Injury Disrupts Phosphatidylinositol 4,5‐Bisphosphate Metabolism Causing Impaired Inward Rectifier Potassium Channel Function

Severe trauma, including traumatic brain injury (TBI), can result in several deleterious outcomes including coagulopathy, multi‐organ failure and death. We previously showed that TBI in rodents causes uncoupling of mesenteric artery (MA) nitric oxide production, resulting in excessive reactive oxygen species (ROS) generation and impaired endothelial‐dependent vasodilation. The inward‐rectifier potassium channel (KIR) is an essential component of agonist‐ and flow‐induced vasodilation and signal transduction in mesenteric arteries (MAs); however, its function following trauma has not been assessed. Phosphatidylinositol 4,5‐bisphosphate (PIP2) is a membrane phospholipid which is required for normal KIR channel function in MAs. We hypothesized that oxidative stress, specifically hydrogen peroxide (H2O2), was driving down PIP2 levels, causing KIR channelopathies. We studied male rats 24 hours after fluid percussion TBI, compared to controls. Plasma oxidative‐reduction potential (ORP), an indicator of oxidative stress, was significantly elevated in TBI rats compared to controls. In TBI but not control plasma, catalase application significantly reduced the ORP signal to control values, suggesting that H2O2 contributes to global oxidative stress following TBI. We then measured KIR currents in isolated endothelial cells (ECs) from MAs, using the whole‐cell patch‐clamp technique from control and TBI rats. KIR channel currents were significantly diminished in ECs from TBI rats compared to controls. KIR currents in ECs from TBI MAs were rescued with the addition of PIP2 to the pipette solution when compared to untreated TBI cells, and showed no difference from their control, treated counterparts, implying depletion of PIP2 following TBI is a contributing factor to KIR channel dysfunction. Next, diameter responses of arteries using pressure myography were measured. In control arteries, increasing concentrations of extracellular potassium from 6 to 10 mM K+ caused dilations; concentrations between 14 to 60 mM K+ caused constrictions. Dilations to 10 mM K+ were significantly reduced following TBI, consistent with an impaired KIR channel response. Next, basal KIR function was assessed using the KIR and KIR2.1 antagonists BaCl2 (100 μM) and ML 133 (20 μM). Following TBI, arteries constricted significantly less to both BaCl2 and ML 133. Furthermore, dilations to 10 mM K+ and constrictions to ML 133 were restored in TBI arteries in the presence of catalase. Lastly, To further quantify cellular pathology following TBI, the metabolomic phenotype was measured by liquid chromatography‐mass spectrometry (LC‐MS) in plasma obtained from control and TBI rats. Rats exhibited a profound metabolopathy following TBI; within the lipidome: arachidonate metabolism, glycerophospholipid synthesis, and fatty acid mobilization were significantly altered. These data suggest H2O2 production serves an antagonistic role on KIR channel function through changes in second messenger and lipid signaling pathways in the mesenteric circulation following TBI.Support or Funding InformationThis work was supported by the Totman Medical Research Trust and the National Institute of Health (K08‐GM‐098795 and UM1‐HL‐120877).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased amplitude of inward rectifier K+ currents with advanced age in smooth muscle cells of murine superior epigastric arteries.

Inward rectifier K+ channels (KIR) may contribute to skeletal muscle blood flow regulation and adapt to advanced age. Using mouse abdominal wall superior epigastric arteries (SEAs) from either young (3-6 mo) or old (24-26 mo) male C57BL/6 mice, we investigated whether SEA smooth muscle cells (SMCs) express functional KIR channels and how aging may affect KIR function. Freshly dissected SEAs were either enzymatically dissociated to isolate SMCs for electrophysiological recording (perforated patch) and mRNA expression or used intact for pressure myography. With 5 mM extracellular K+ concentration ([K+]o), exposure of SMCs to the KIR blocker Ba2+ (100 μM) had no significant effect (P > 0.05) on whole cell currents elicited by membrane potentials spanning -120 to -30 mV. Raising [K+]o to 15 mM activated Ba2+-sensitive KIR currents between -120 and -30 mV, which were greater in SMCs from old mice than in SMCs from young mice (P < 0.05). Pressure myography of SEAs revealed that while aging decreased maximum vessel diameter by ~8% (P < 0.05), it had no significant effect on resting diameter, myogenic tone, dilation to 15 mM [K+]o, Ba2+-induced constriction in 5 mM [K+]o, or constriction induced by 15 mM [K+]o in the presence of Ba2+ (P > 0.05). Quantitative RT-PCR revealed SMC expression of KIR2.1 and KIR2.2 mRNA that was not affected by age. Barium-induced constriction of SEAs from young and old mice suggests an integral role for KIR in regulating resting membrane potential and vasomotor tone. Increased functional expression of KIR channels during advanced age may compensate for other age-related changes in SEA function.NEW & NOTEWORTHY Ion channels are integral to blood flow regulation. We found greater functional expression of inward rectifying K+ channels in smooth muscle cells of resistance arteries of mouse skeletal muscle with advanced age. This adaptation to aging may contribute to the maintenance of vasomotor tone and blood flow regulation during exercise.

Uncoupling of neurovascular communication after transient global cerebral ischemia is caused by impaired parenchymal smooth muscle Kir channel function

Transient global cerebral ischemia is often followed by delayed disturbances of cerebral blood flow, contributing to neuronal injury. The pathophysiological processes underlying such disturbances are incompletely understood. Here, using an established model of transient global cerebral ischemia, we identify dramatically impaired neurovascular coupling following ischemia. This impairment results from the loss of functional inward rectifier potassium (KIR) channels in the smooth muscle of parenchymal arterioles. Therapeutic strategies aimed at protecting or restoring cerebrovascular KIR channel function may therefore improve outcomes following ischemia.

Inward rectifier potassium (Kir2.1) channels as end-stage boosters of endothelium-dependent vasodilators.

Increase in endothelial cell (EC) calcium activates calcium-sensitive intermediate and small conductance potassium (IK and SK) channels, thereby causing hyperpolarization and endothelium-dependent vasodilatation. Endothelial cells express inward rectifier potassium (Kir) channels, but their role in endothelium-dependent vasodilatation is not clear. In the mesenteric arteries, only ECs, but not smooth muscle cells, displayed Kir currents that were predominantly mediated by the Kir2.1 isoform. Endothelium-dependent vasodilatations in response to muscarinic receptor, TRPV4 (transient receptor potential vanilloid 4) channel and IK/SK channel agonists were highly attenuated by Kir channel inhibitors and by Kir2.1 channel knockdown. These results point to EC Kir channels as amplifiers of vasodilatation in response to increases in EC calcium and IK/SK channel activation and suggest that EC Kir channels could be targeted to treat endothelial dysfunction, which is a hallmark of vascular disorders. Endothelium-dependent vasodilators, such as acetylcholine, increase intracellular Ca(2+) through activation of transient receptor potential vanilloid 4 (TRPV4) channels in the plasma membrane and inositol trisphosphate receptors in the endoplasmic reticulum, leading to stimulation of Ca(2+) -sensitive intermediate and small conductance K(+) (IK and SK, respectively) channels. Although strong inward rectifier K(+) (Kir) channels have been reported in the native endothelial cells (ECs) their role in EC-dependent vasodilatation is not clear. Here, we test the idea that Kir channels boost the EC-dependent vasodilatation of resistance-sized arteries. We show that ECs, but not smooth muscle cells, of small mesenteric arteries have Kir currents, which are substantially reduced in EC-specific Kir2.1 knockdown (EC-Kir2.1(-/-) ) mice. Elevation of extracellular K(+) to 14mm caused vasodilatation of pressurized arteries, which was prevented by endothelial denudation and Kir channel inhibitors (Ba(2+) , ML-133) or in the arteries from EC-Kir2.1(-/-) mice. Potassium-induced dilatations were unaffected by inhibitors of TRPV4, IK and SK channels. The Kir channel blocker, Ba(2+) , did not affect currents through TRPV4, IK or SK channels. Endothelial cell-dependent vasodilatations in response to activation of muscarinic receptors, TRPV4 channels or IK/SK channels were reduced, but not eliminated, by Kir channel inhibitors or EC-Kir2.1(-/-) . In angiotensinII-induced hypertension, the Kir channel function was not altered, although the endothelium-dependent vasodilatation was severely impaired. Our results support the concept that EC Kir2 channels boost vasodilatory signals that are generated by Ca(2+) -dependent activation of IK and SK channels.

Molecular Dynamics Simulations of Kir2.2-Cholesterol Interactions

Inwardly rectifying potassium (Kir) channels form a major family of potassium channels and have been shown to play a critical role in setting resting membrane potential, regulating potassium homeostasis, and modulating membrane excitability. Cholesterol, which forms a major component of mammalian cell membranes, has been shown in the past to suppress Kir family channel function in high concentrations and is linked to a number of pathological conditions. Recently, it has been proposed that cholesterol binds to specific non-annular sites between the transmembrane domains of the Kir protein, and that this bonding helps to stabilize a closed conformation for the channel. In order to better understand this interaction, we have taken a computational approach which utilizes coarse-grained molecular dynamics simulations - employing the MARTINI force field - to further investigate the spatial and temporal mechanics of cholesterol binding. In addition to the mechanics of binding, this approach has allowed us to more closely examine the physiological effects of cholesterol binding on Kir channel function in a more physiologically relevant membrane environment. Through this we have been able to examine a greater range of binding effects, such as binding time, binding instances, changes in channel conformation, and ion flux through the channel.

Hyperglycemia reduces functional expression of astrocytic Kir4.1 channels and glial glutamate uptake

Diabetics are at risk for a number of serious health complications including an increased incidence of epilepsy and poorer recovery after ischemic stroke. Astrocytes play a critical role in protecting neurons by maintaining extracellular homeostasis and preventing neurotoxicity through glutamate uptake and potassium buffering. These functions are aided by the presence of potassium channels, such as Kir4.1 inwardly rectifying potassium channels, in the membranes of astrocytic glial cells. The purpose of the present study was to determine if hyperglycemia alters Kir4.1 potassium channel expression and homeostatic functions of astrocytes. We used q-PCR, Western blot, patch-clamp electrophysiology studying voltage and potassium step responses and a colorimetric glutamate clearance assay to assess Kir4.1 channel levels and homeostatic functions of rat astrocytes grown in normal and high glucose conditions. We found that astrocytes grown in high glucose (25mM) had an approximately 50% reduction in Kir4.1 mRNA and protein expression as compared with those grown in normal glucose (5mM). These reductions occurred within 4–7days of exposure to hyperglycemia, whereas reversal occurred between 7 and 14days after return to normal glucose. The decrease in functional Kir channels in the astrocytic membrane was confirmed using barium to block Kir channels. In the presence of 100-μM barium, the currents recorded from astrocytes in response to voltage steps were reduced by 45%. Furthermore, inward currents induced by stepping extracellular [K+]o from 3 to 10mM (reflecting potassium uptake) were 50% reduced in astrocytes grown in high glucose. In addition, glutamate clearance by astrocytes grown in high glucose was significantly impaired. Taken together, our results suggest that down-regulation of astrocytic Kir4.1 channels by elevated glucose may contribute to the underlying pathophysiology of diabetes-induced CNS disorders and contribute to the poor prognosis after stroke.

Inward rectifier potassium currents in mammalian skeletal muscle fibres.

Inward rectifying potassium (Kir) channels play a central role in maintaining the resting membrane potential of skeletal muscle fibres. Nevertheless their role has been poorly studied in mammalian muscles. Immunohistochemical and transgenic expression were used to assess the molecular identity and subcellular localization of Kir channel isoforms. We found that Kir2.1 and Kir2.2 channels were targeted to both the surface and the transverse tubular system membrane (TTS) compartments and that both isoforms can be overexpressed up to 3-fold 2 weeks after transfection. Inward rectifying currents (IKir) had the canonical features of quasi-instantaneous activation, strong inward rectification, depended on the external [K(+)], and could be blocked by Ba(2+) or Rb(+). In addition, IKir records show notable decays during large 100 ms hyperpolarizing pulses. Most of these properties were recapitulated by model simulations of the electrical properties of the muscle fibre as long as Kir channels were assumed to be present in the TTS. The model also simultaneously predicted the characteristics of membrane potential changes of the TTS, as reported optically by a fluorescent potentiometric dye. The activation of IKir by large hyperpolarizations resulted in significant attenuation of the optical signals with respect to the expectation for equal magnitude depolarizations; blocking IKir with Ba(2+) (or Rb(+)) eliminated this attenuation. The experimental data, including the kinetic properties of IKir and TTS voltage records, and the voltage dependence of peak IKir, while measured at widely dissimilar bulk [K(+)] (96 and 24 mm), were closely predicted by assuming Kir permeability (PKir) values of ∼5.5 × 10(-6 ) cm s(-1) and equal distribution of Kir channels at the surface and TTS membranes. The decay of IKir records and the simultaneous increase in TTS voltage changes were mostly explained by K(+) depletion from the TTS lumen. Most importantly, aside from allowing an accurate estimation of most of the properties of IKir in skeletal muscle fibres, the model demonstrates that a substantial proportion of IKir (>70%) arises from the TTS. Overall, our work emphasizes that measured intrinsic properties (inward rectification and external [K] dependence) and localization of Kir channels in the TTS membranes are ideally suited for re-capturing potassium ions from the TTS lumen during, and immediately after, repetitive stimulation under physiological conditions.