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Aim: Long QT syndrome (LQTS) and catecholaminergic polymorphism ventricular tachycardia (CPVT) are inherited cardiac disorders often caused by mutations in ion channels. These arrhythmia syndromes have recently been associated with calmodulin (CaM) variants. Here, we investigate the impact of the arrhythmogenic variants D131E and Q135P on CaM's structure-function relationship. Our study focuses on the L-type calcium channel Ca v 1.2, a crucial component of the ventricular action potential and excitation-contraction coupling., Methods: We used circular dichroism (CD), 1 H- 15 N HSQC NMR, and trypsin digestion to determine the structural and stability properties of CaM variants. The affinity of CaM for Ca 2+ and interaction of Ca 2+ /CaM with Ca v 1.2 (IQ and NSCaTE domains) were investigated using intrinsic tyrosine fluorescence and isothermal titration calorimetry (ITC), respectively. The effect of CaM variants of Ca v 1.2 activity was determined using HEK293-Ca v 1.2 cells (B'SYS) and whole-cell patch-clamp electrophysiology., Results: Using a combination of protein biophysics and structural biology, we show that the disease-associated mutations D131E and Q135P mutations alter apo/CaM structure and stability. In the Ca 2+ -bound state, D131E and Q135P exhibited reduced Ca 2+ binding affinity, significant structural changes, and altered interaction with Ca v 1.2 domains (increased affinity for Ca v 1.2-IQ and decreased affinity for Ca v 1.2-NSCaTE). We show that the mutations dramatically impair Ca 2+ -dependent inactivation (CDI) of Ca v 1.2, which would contribute to abnormal Ca 2+ influx, leading to disrupted Ca 2+ handling, characteristic of cardiac arrhythmia syndromes., Conclusions: These findings provide insights into the molecular mechanisms behind arrhythmia caused by calmodulin mutations, contributing to our understanding of cardiac syndromes at a molecular and cellular level., (© 2025 The Author(s). Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Background and Purpose: The canonical K ir 6.2/SUR2A ventricular K ATP channel is highly ATP-sensitive and remains closed under normal physiological conditions. These channels activate only when prolonged metabolic compromise causes significant ATP depletion and then shortens the action potential to reduce contractile activity. Pharmacological activation of K ATP channels is cardioprotective, but physiologically, it is difficult to understand how these channels protect the heart if they only open under extreme metabolic stress. The presence of a second K ATP channel population could help explain this. Here, we characterise the biophysical and pharmacological behaviours of a constitutively active K ir 6.1-containing K ATP channel in ventricular cardiomyocytes., Experimental Approach: Patch-clamp recordings from rat ventricular myocytes in combination with well-defined pharmacological modulators was used to characterise these newly identified K + channels. Action potential recording, calcium (Fluo-4) fluorescence measurements and video edge detection of contractile function were used to assess functional consequences of channel modulation., Key Results: Our data show a ventricular K + conductance whose biophysical characteristics and response to pharmacological modulation were consistent with K ir 6.1-containing channels. These K ir 6.1-containing channels lack the ATP-sensitivity of the canonical channels and are constitutively active., Conclusion and Implications: We conclude there are two functionally distinct populations of ventricular K ATP channels: constitutively active K ir 6.1-containing channels that play an important role in fine-tuning the action potential and K ir 6.2/SUR2A channels that activate with prolonged ischaemia to impart late-stage protection against catastrophic ATP depletion. Further research is required to determine whether K ir 6.1 is an overlooked target in Comprehensive in vitro Proarrhythmia Assay (CiPA) cardiac safety screens., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Calmodulin (CaM) is a highly conserved mediator of calcium (Ca 2+ )-dependent signalling and modulates various cardiac ion channels. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS). LQTS patients display prolonged ventricular recovery times (QT interval), increasing their risk of incurring life-threatening arrhythmic events. Loss-of-function mutations to Kv7.1 (which drives the slow delayed rectifier potassium current, IKs, a key ventricular repolarising current) are the largest contributor to congenital LQTS (>50% of cases). CaM modulates Kv7.1 to produce a Ca 2+ -sensitive IKs, but little is known about the consequences of LQTS-associated CaM mutations on Kv7.1 function. Here, we present novel data characterising the biophysical and modulatory properties of three LQTS-associated CaM variants (D95V, N97I and D131H). We showed that mutations induced structural alterations in CaM and reduced affinity for Kv7.1, when compared with wild-type (WT). Using HEK293T cells expressing Kv7.1 channel subunits (KCNQ1/KCNE1) and patch-clamp electrophysiology, we demonstrated that LQTS-associated CaM variants reduced current density at systolic Ca 2+ concentrations (1 μm), revealing a direct QT-prolonging modulatory effect. Our data highlight for the first time that LQTS-associated perturbations to CaM's structure impede complex formation with Kv7.1 and subsequently result in reduced IKs. This provides a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype. KEY POINTS: Calmodulin (CaM) is a ubiquitous, highly conserved calcium (Ca 2+ ) sensor playing a key role in cardiac muscle contraction. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS), a life-threatening cardiac arrhythmia syndrome. LQTS-associated CaM variants (D95V, N97I and D131H) induced structural alterations, altered binding to Kv7.1 and reduced IKs. Our data provide a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Skin is the largest organ in the human body with ∼95% of its surface made up of keratinocytes. These cells maintain a healthy skin barrier through regulated differentiation driven by Ca 2+ -transcriptional coupling. Many important skin conditions arise from disruption of this process although not all stages are fully understood. We know that elevated extracellular Ca 2+ at the skin surface is detected by keratinocyte Gα q -coupled receptors that signal to empty endoplasmic reticulum Ca 2+ stores. Orai channel store-operated Ca 2+ entry (SOCE) and Ca 2+ influx via "canonical" transient receptor potential (TRPC)-composed channels then activates transcription factors that drive differentiation. While STIM-mediated activation of Orai channels following store depletion is well defined, how TRPC channels are activated is less clear. Multiple modes of TRPC channel activation have been proposed, including 1) independent TRPC activation by STIM, 2) formation of Orai-TRPC-STIM complexes, and 3) the insertion of constitutively-active TRPC channels into the membrane during SOCE. To help distinguish between these models, we used high-resolution microscopy of intact keratinocyte (HaCaT) cells and immunogold transmission electron microscopy (TEM) of HaCaT plasma membrane sheets. Our data shows no evidence of significant insertion of Orai1 or TRPC subunits into the membrane during SOCE. Analysis of transmission electron microscopy data shows that during store-depletion and SOCE, Orai1 and TRPC subunits form separate membrane-localized clusters that migrate towards each other. This clustering of TRPC channel subunits in keratinocytes may support the formation of TRPC-STIM interactions at ER-plasma membrane junctions that are distinct from Orai-STIM junctions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Manning, Barrett-Jolley, Evans and Dart.)

Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca 2+ -sensing protein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Ca v 1.2 Ca 2+ /CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM's Ca 2+ -binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Ca v 1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Ca v 1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ., Competing Interests: Conflict of interest The authors declare no competing or financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Multiple organ injury and dysfunction often occurs in acute critical illness and adversely affects survival. However, in patients who survive, organ function usually recovers without permanent damage. It is, therefore, likely that there are reversible mechanisms, but this is poorly understood in the pathogenesis of multiple organ dysfunction syndrome (MODS)., Aims: Based on our knowledge of extracellular histones and pneumolysin, as endogenous and exogenous pore-forming toxins, respectively, here we clarify if the extent of cell membrane disruption and recovery is important in MODS., Methods: This is a combination of retrospective clinical studies of a cohort of 98 patients from an intensive care unit (ICU) in a tertiary hospital, with interventional animal models and laboratory investigation., Results: In patients without septic shock and/or disseminate intravascular coagulation (DIC), circulating histones also strongly correlated with sequential organ failure assessment (SOFA) scores, suggesting their pore-forming property might play an important role. In vivo, histones or pneumolysin infusion similarly caused significant elevation of cell damage markers and multiple organ injury. In trauma and sepsis models, circulating histones strongly correlated with these markers, and anti-histone reagents significantly reduced their release. Comparison of pneumolysin deletion and its parental strain-induced sepsis mouse model showed that pneumolysin was not essential for sepsis development, but enhanced multiple organ damage and reduced survival time. In vitro, histones and pneumolysin treatment disrupt cell membrane integrity, resulting in changes in whole-cell currents and elevated intracellular Ca 2+ to lead to Ca 2+ overload. Cell-specific damage markers, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and cardiac troponin I (cTnI), were released from damaged cells. Once toxins were removed, cell membrane damage could be rapidly repaired and cellular function recovered., Conclusion: This work has confirmed the importance of pore-forming toxins in the development of MODS and proposed a potential mechanism to explain the reversibility of MODS. This may form the foundation for the development of effective therapies., Competing Interests: The authors declare no conflict of interest.

The skin is a complex organ that acts as a protective layer against the external environment. It protects the internal tissues from harmful agents, dehydration, ultraviolet radiation and physical injury as well as conferring thermoregulatory control, sensation, immunological surveillance and various biochemical functions. The diverse cell types that make up the skin include 1) keratinocytes, which form the bulk of the protective outer layer; 2) melanocytes, which protect the body from ultraviolet radiation by secreting the pigment melanin; and 3) cells that form the secretory appendages: eccrine and apocrine sweat glands, and the sebaceous gland. Emerging evidence suggests that store-operated Ca 2+ entry (SOCE), whereby depletion of intracellular Ca 2+ stores triggers Ca 2+ influx across the plasma membrane, is central to the normal physiology of these cells and thus skin function. Numerous skin pathologies including dermatitis, anhidrotic ectodermal dysplasia, hyperhidrosis, hair loss and cancer are now linked to dysfunction in SOCE proteins. Principal amongst these are the stromal interaction molecules (STIMs) that sense Ca 2+ depletion and Orai channels that mediate Ca 2+ influx. In this review, the roles of STIM, Orai and other store-operated channels are discussed in the context of keratinocyte differentiation, melanogenesis, and eccrine sweat secretion. We explore not only STIM1-Orai1 as drivers of SOCE, but also independent actions of STIM, and emerging signal cascades stemming from their activities. Roles are discussed for the elusive transient receptor potential canonical channel (TRPC) complex in keratinocytes, Orai channels in Ca 2+ -cyclic AMP signal crosstalk in melanocytes, and Orai isoforms in eccrine sweat gland secretion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Manning, Dart and Evans.)

ATP-sensitive potassium (K ATP ) channels couple cellular metabolism to excitability, making them ideal candidate sensors for hypoxic vasodilation. However, it is still unknown whether cellular nucleotide levels are affected sufficiently to activate vascular K ATP channels during hypoxia. To address this fundamental issue, we measured changes in the intracellular ATP:ADP ratio using the biosensors Perceval/PercevalHR, and membrane potential using the fluorescent probe DiBAC 4 (3) in human coronary artery smooth muscle cells (HCASMCs). ATP:ADP ratio was significantly reduced by exposure to hypoxia. Application of metabolic inhibitors for oxidative phosphorylation also reduced ATP:ADP ratio. Hyperpolarization caused by inhibiting oxidative phosphorylation was blocked by either 10 µM glibenclamide or 60 mM K + . Hyperpolarization caused by hypoxia was abolished by 60 mM K + but not by individual K + channel inhibitors. Taken together, these results suggest hypoxia causes hyperpolarization in part by modulating K + channels in SMCs., Competing Interests: The authors declare there are no competing interests., (©2020 Yang et al.)

Single-molecule research techniques such as patch-clamp electrophysiology deliver unique biological insight by capturing the movement of individual proteins in real time, unobscured by whole-cell ensemble averaging. The critical first step in analysis is event detection, so called "idealisation", where noisy raw data are turned into discrete records of protein movement. To date there have been practical limitations in patch-clamp data idealisation; high quality idealisation is typically laborious and becomes infeasible and subjective with complex biological data containing many distinct native single-ion channel proteins gating simultaneously. Here, we show a deep learning model based on convolutional neural networks and long short-term memory architecture can automatically idealise complex single molecule activity more accurately and faster than traditional methods. There are no parameters to set; baseline, channel amplitude or numbers of channels for example. We believe this approach could revolutionise the unsupervised automatic detection of single-molecule transition events in the future.

Ca 2+ -transcription coupling controls gene expression patterns that define vascular smooth muscle cell (VSMC) phenotype. Although not well understood this allows normally contractile VSMCs to become proliferative following vessel injury, a process essential for repair but which also contributes to vascular remodelling, atherogenesis and restenosis. Here we show that the Ca 2+ /HCO 3 - -sensitive enzyme, soluble adenylyl cyclase (sAC), links Ca 2+ influx in human coronary artery smooth muscle cells (hCASMCs) to 3',5'-cyclic adenosine monophosphate (cAMP) generation and phosphorylation of the transcription factor Ca 2+ /cAMP response element binding protein (CREB). Store-operated Ca 2+ entry (SOCE) into hCASMCs expressing the FRET-based cAMP biosensor H187 induced a rise in cAMP that mirrored cytosolic [Ca 2+ ]. SOCE also activated the cAMP effector, protein kinase A (PKA), as determined by the PKA reporter, AKAR4-NES, and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and CREB. Transmembrane adenylyl cyclase inhibition had no effect on the SOCE-induced rise in cAMP, while sAC inhibition abolished SOCE-generated cAMP and significantly reduced SOCE-induced VASP and CREB phosphorylation. This suggests that SOCE in hCASMCs activates sAC which in turn activates the cAMP/PKA/CREB axis. sAC, which is insensitive to G-protein modulation but responsive to Ca 2+ , pH and ATP, may therefore act as an overlooked regulatory node in vascular Ca 2+ -transcription coupling.

Key Points: The Ca 2+ and redox-sensing enzyme Ca 2+ /calmodulin-dependent kinase 2 (CaMKII) is a crucial and well-established signalling molecule in the heart and brain. In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca 2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII. The vasodilator-induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage-dependent Ca 2+ influx. How Epac activates STOCs is unknown. In the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII. To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation., Abstract: Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)-sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca 2+ sparks activate plasma membrane Ca 2+ -activated K + (BK Ca ) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage-dependent Ca 2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5-cyclic monophosphate-AM (8-pCPT-AM) induces autophosphorylation (activation) of calcium/calmodulin-dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8-pCPT-AM-induced increases in STOC activity. Epac-induced CaMKII activation is probably initiated by inositol 1,4,5-trisphosphate (IP 3 )-mobilized Ca 2+ : 8-pCPT-AM fails to induce CaMKII activation following intracellular Ca 2+ store depletion and inhibition of IP 3 receptors blocks both 8-pCPT-AM-mediated CaMKII phosphorylation and STOC activity. 8-pCPT-AM does not directly activate BK Ca channels, but STOCs cannot be generated by 8-pCPT-AM in the presence of ryanodine. Furthermore, exposure to 8-pCPT-AM significantly slows the initial rate of [Ca 2+ ] i rise induced by the RyR activator caffeine without significantly affecting the caffeine-induced Ca 2+ transient amplitude, a measure of Ca 2+ store content. We conclude that Epac-mediated STOC activity (i) occurs via activation of CaMKII and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge, this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca 2+ and redox-sensing enzyme in the regulation of vascular tone and blood flow., (© 2017 The Authors The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter.

Potassium (K(+)) channels, with their diversity, often tissue-defined distribution, and critical role in controlling cellular excitability, have long held promise of being important drug targets for the treatment of dysrhythmias in the heart and abnormal neuronal activity within the brain. With the exception of drugs that target one particular class, ATP-sensitive K(+) (KATP) channels, very few selective K(+) channel activators or inhibitors are currently licensed for clinical use in cardiovascular and neurological disease. Here we review what a range of human genetic disorders have told us about the role of specific K(+) channel subunits, explore the potential of activators and inhibitors of specific channel populations as a therapeutic strategy, and discuss possible reasons for the difficulty in designing clinically relevant K(+) channel modulators., (© 2015 Society for Laboratory Automation and Screening.)

The second messenger cAMP plays a central role in mediating vascular smooth muscle relaxation in response to vasoactive transmitters and in strengthening endothelial cell-cell junctions that regulate the movement of solutes, cells and macromolecules between the blood and the surrounding tissue. The vasculature expresses three cAMP effector proteins: PKA (protein kinase A), CNG (cyclic-nucleotide-gated) ion channels, and the most recently discovered Epacs (exchange proteins directly activated by cAMP). Epacs are a family of GEFs (guanine-nucleotide-exchange factors) for the small Ras-related GTPases Rap1 and Rap2, and are being increasingly implicated as important mediators of cAMP signalling, both in their own right and in parallel with the prototypical cAMP target PKA. In the present paper, we review what is currently known about the role of Epac within blood vessels, particularly with regard to the regulation of vascular tone, endothelial barrier function and inflammation.

Vasodilator-induced elevation of intracellular cyclic AMP (cAMP) is a central mechanism governing arterial relaxation but is incompletely understood due to the diversity of cAMP effectors. Here we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. In myography experiments, the Epac-selective cAMP analogue 8-pCPT-2-O-Me-cAMP-AM (5 μM, subsequently referred to as 8-pCPT-AM) elicited a 77.6 ± 7.1% relaxation of phenylephrine-contracted arteries over a 5 min period (mean ± SEM; n = 6). 8-pCPT-AM induced only a 16.7 ± 2.4% relaxation in arteries pre-contracted with high extracellular K(+) over the same time period (n = 10), suggesting that some of Epac's relaxant effect relies upon vascular cell hyperpolarization. This involves Ca(2+)-sensitive, large-conductance K(+) (BK(Ca)) channel opening as iberiotoxin (100 nM) significantly reduced the ability of 8-pCPT-AM to reverse phenylephrine-induced contraction (arteries relaxed by only 35.0 ± 8.5% over a 5 min exposure to 8-pCPT-AM, n = 5; P < 0.05). 8-pCPT-AM increased Ca(2+) spark frequency in Fluo-4-AM-loaded mesenteric myocytes from 0.045 ± 0.008 to 0.103 ± 0.022 sparks s(-1) μm(-1) (P < 0.05) and reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72 s(-1)) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n = 7; P < 0.05). 8-pCPT-AM-activated STOCs were sensitive to iberiotoxin (100 nM) and to ryanodine (30 μM). Current clamp recordings of isolated myocytes showed a 7.9 ± 1.0 mV (n = 10) hyperpolarization in response to 8-pCPT-AM that was sensitive to iberiotoxin (n = 5). Endothelial disruption suppressed 8-pCPT-AM-mediated relaxation in phenylephrine-contracted arteries (24.8 ± 4.9% relaxation after 5 min of exposure, n = 5; P < 0.05), as did apamin and TRAM-34, blockers of Ca(2+)-sensitive, small- and intermediate-conductance K(+) (SK(Ca) and IK(Ca)) channels, respectively, and N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS). In Fluo-4-AM-loaded mesenteric endothelial cells, 8-pCPT-AM induced a sustained increase in global Ca(2+). Our data suggest that Epac hyperpolarizes smooth muscle by (1) increasing localized Ca(2+) release from ryanodine receptors (Ca(2+) sparks) to activate BK(Ca) channels, and (2) endothelial-dependent mechanisms involving the activation of SK(Ca)/IK(Ca) channels and NOS. Epac-mediated smooth muscle hyperpolarization will limit Ca(2+) entry via voltage-sensitive Ca(2+) channels and represents a novel mechanism of arterial relaxation.

C-reactive protein (CRP) is an acute-phase protein that plays an important defensive role in innate immunity against bacterial infection, but it is also upregulated in many noninfectious diseases. The generic function of this highly conserved molecule in diseases that range from infection, inflammation, trauma, and malignancy is not well understood. In this article, we demonstrate that CRP defends the human body against the toxicity of histones released into the circulation after extensive cell death. In vitro, CRP significantly alleviates histone-induced endothelial cell damage, permeability increase, and platelet aggregation. In vivo, CRP rescues mice challenged with lethal doses of histones by inhibiting endothelial damage, vascular permeability, and coagulation activation, as reflected by significant reductions in lung edema, hemorrhage, and thrombosis. In patients, elevation of CRP significantly increases the capacity to neutralize extracellular histones in the circulation. We have also confirmed that CRP interacts with individual histones in vitro and forms CRP-histone complexes in serum from patients with both elevated CRP and histones. CRP is able to compete with phospholipid-containing liposomes for the binding to histones. This explains how CRP prevents histones from integrating into cell membranes, which would otherwise induce calcium influx as the major mechanism of cytotoxicity caused by extracellular histones. Because histone elevation occurs in the acute phase of numerous critical illnesses associated with extensive cell death, CRP detoxification of circulating histones would be a generic host defense mechanism in humans.

Rationale: Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology., Objectives: To investigate the pathological roles of circulating histones in trauma-induced lung injury., Methods: Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause-effect relationship was studied using cells and mouse models., Measurements and Main Results: In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality., Conclusions: This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival outcomes in patients.

The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a key role in the control of eukaryotic cellular activity. The enzymology of PK-A in the free-living nematode, Caenorhabditis elegans is deceptively simple. Single genes encode the catalytic (C) subunit (kin-1), the regulatory (R) subunit (kin-2) and an A-kinase anchor protein (AKAP) (aka-1); nonetheless, PK-A is able to facilitate a comprehensive array of cAMP-mediated processes in this model multicellular organism. We have previously demonstrated that, in C. elegans, as many as 12 different isoforms of the C-subunit arise as a consequence of alternative splicing strategies. Here, we report the occurrence of transcripts encoding novel isoforms of the PK-A R-subunit in C. elegans. In place of exons 1 and 2, these transcripts include coding sequences from novel B or Q exons directly linked to exon 3, thereby generating isoforms with novel N-termini. R-subunits containing an exon B-encoded N-terminal polypeptide sequence were detected in extracts prepared from mixed populations of C. elegans. Of note is the observation that R-subunit isoforms containing exon B- or exon Q-encoded polypeptide sequences lack the dimerisation/docking domains conventionally seen in R-subunits. This means that they are unlikely to participate in the formation of tetrameric PK-A holoenzymes and, additionally, they are unlikely to interact with AKAP(s). It is therefore possible that, in C. elegans, in addition to tetrameric (R(2)C(2)) PK-A holoenzymes, there is also a sub-population of dimeric (RC) PK-A enzymes that are not tethered by AKAPs. Furthermore, inspection of the N-terminal sequence encoded by exon B suggests that this isoform is a likely target for N-myristoylation. Although unusual, a number of similarly N-myristoylatable R-subunits, from a range of different species, are present in the databases, suggesting that this may be a more generally observed feature of R-subunit structure. The occurrence of R-subunit isoforms, without dimerisation/docking domains (with or without N-myristoylatable N-termini) in other species would suggest that the control of PK-A activity may be more complex than hitherto thought., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Chondrocytes are the resident cells of cartilage, which synthesize and maintain the extracellular matrix. The range of known potassium channels expressed by these unique cells is continually increasing. Since chondrocytes are non-excitable, and do not need to be repolarized following action potentials, the function of potassium channels in these cells has, until recently, remained completely unknown. However, recent advances in both traditional physiology and "omic" technologies have enhanced our knowledge and understanding of the chondrocyte channelome. A large number of potassium channels have been identified and a number of putative, but credible, functions have been proposed. Members of each of the potassium channel sub-families (calcium activated, inward rectifier, voltage-gated and tandem pore) have all been identified. Mechanotransduction, cell volume regulation, apoptosis and chondrogenesis all appear to involve potassium channels. Since evidence suggests that potassium channel gene transcription is altered in osteoarthritis, future studies are needed that investigate potassium channels as potential cellular biomarkers and therapeutic targets for treatment of degenerative joint conditions.

Rosiglitazone, the thiazolidinedione class anti-diabetic withdrawn from Europe in 2010 amid reports of adverse cardiovascular effects, is revealed by Yu et al. in this issue of the British Journal of Pharmacology to be a selective blocker of ATP-sensitive potassium (K(ATP) ) channels. This seems little cause for excitement given that the closure of pancreatic K(ATP) channels is integral to insulin secretion; and sulphonylureas, which inhibit K(ATP) channels, are widely used to treat type II diabetes. However, rosiglitazone, whose primary targets are nuclear transcription factors that regulate genes involved in lipid metabolism, blocks K(ATP) channels by a novel mechanism different to that of the sulphonylureas and has a worrying preference for blood flow-regulating vascular K(ATP) channels. Identification of a new molecule that modulates K(ATP) channel gating will not only tell us more about how these complex metabolic sensors work but also raises questions as to whether rosiglitazone suppresses the cardiovascular system's ability to cope with metabolic stress - a claim that has dogged the sulphonylureas for many years., (© 2012 The Author. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Caveolins are coat proteins of caveolae, small flask-shaped pits of the plasma membranes of most cells. Aside from roles in caveolae formation, caveolins recruit, retain and regulate many caveolae-associated signalling molecules. Caveolin-protein interactions are commonly considered to occur between a ∼20 amino acid region within caveolin, the caveolin scaffolding domain (CSD), and an aromatic-rich caveolin binding motif (CBM) on the binding partner (фXфXXXXф, фXXXXфXXф or фXфXXXXфXXф, where ф is an aromatic and X an unspecified amino acid). The CBM resembles a typical linear motif--a short, simple sequence independently evolved many times in different proteins for a specific function. Here we exploit recent improvements in bioinformatics tools and in our understanding of linear motifs to critically examine the role of CBMs in caveolin interactions. We find that sequences conforming to the CBM occur in 30% of human proteins, but find no evidence for their statistical enrichment in the caveolin interactome. Furthermore, sequence- and structure-based considerations suggest that CBMs do not have characteristics commonly associated with true interaction motifs. Analysis of the relative solvent accessible area of putative CBMs shows that the majority of their aromatic residues are buried within the protein and are thus unlikely to interact directly with caveolin, but may instead be important for protein structural stability. Together, these findings suggest that the canonical CBM may not be a common characteristic of caveolin-target interactions and that interfaces between caveolin and targets may be more structurally diverse than presently appreciated.

Many cell types have significant negative resting membrane potentials (RMPs) resulting from the activity of potassium-selective and chloride-selective ion channels. In excitable cells, such as neurones, rapid changes in membrane permeability underlie the generation of action potentials. Chondrocytes have less negative RMPs and the role of the RMP is not clear. Here we examine the basis of the chondrocyte RMP and possible physiological benefits. We demonstrate that maintenance of the chondrocyte RMP involves gadolinium-sensitive cation channels. Pharmacological inhibition of these channels causes the RMP to become more negative (100 µM gadolinium: ΔV(m)  = -30 ± 4 mV). Analysis of the gadolinium-sensitive conductance reveals a high permeability to calcium ions (PCa/PNa ≈80) with little selectivity between monovalent ions; similar to that reported elsewhere for TRPV5. Detection of TRPV5 by PCR and immunohistochemistry and the sensitivity of the RMP to the TRPV5 inhibitor econazole (ΔV(m)  = -18 ± 3 mV) suggests that the RMP may be, in part, controlled by TRPV5. We investigated the physiological advantage of the relatively positive RMP using a mathematical model in which membrane stretch activates potassium channels allowing potassium efflux to oppose osmotic water uptake. At very negative RMP potassium efflux is negligible, but at more positive RMP it is sufficient to limit volume increase. In support of our model, cells clamped at -80 mV and challenged with a reduced osmotic potential swelled approximately twice as much as cells at +10 mV. The positive RMP may be a protective adaptation that allows chondrocytes to respond to the dramatic osmotic changes, with minimal changes in cell volume., (Copyright © 2011 Wiley-Liss, Inc.)

The paraventricular nucleus (PVN) of the hypothalamus has been described as the "autonomic master controller". It co-ordinates critical physiological responses through control of the hypothalamic-pituitary-adrenal (HPA)-axis, and by modulation of the sympathetic and parasympathetic branches of the central nervous system. The PVN comprises several anatomical subdivisions, including the parvocellular/ mediocellular subdivision, which contains neurones projecting to the medulla and spinal cord. Consensus indicates that output from spinally-projecting sympathetic pre-autonomic neurones (SPANs) increases blood pressure and heart rate, and dysfunction of these neurones has been directly linked to elevated sympathetic activity during heart failure. The influence of spinally-projecting SPANs on cardiovascular function high-lights their potential as targets for future therapeutic drug development. Recent studies have demonstrated pharmacological control of these spinally-projecting SPANs with glutamate, GABA, nitric oxide, neuroactive steroids and a number of neuropeptides (including angiotensin, substance P, and corticotrophin-releasing factor). The underlying mechanism of control appears to be a state of tonic inhibition by GABA, which is then strengthened or relieved by the action of other modulators. The physiological function of spinally-projecting SPANs has been subject to some debate, and they may be involved in physiological stress responses, blood volume regulation, glucose regulation, thermoregulation and/or circadian rhythms. This review describes the pharmacology of PVN spinally-projecting SPANs and discusses their likely roles in cardiovascular control.

Many types of ion channel localize to cholesterol and sphingolipid-enriched regions of the plasma membrane known as lipid microdomains or 'rafts'. The precise physiological role of these unique lipid microenvironments remains elusive due largely to difficulties associated with studying these potentially extremely small and dynamic domains. Nevertheless, increasing evidence suggests that membrane rafts regulate channel function in a number of different ways. Raft-enriched lipids such as cholesterol and sphingolipids exert effects on channel activity either through direct protein-lipid interactions or by influencing the physical properties of the bilayer. Rafts also appear to selectively recruit interacting signalling molecules to generate subcellular compartments that may be important for efficient and selective signal transduction. Direct interaction with raft-associated scaffold proteins such as caveolin can also influence channel function by altering gating kinetics or by affecting trafficking and surface expression. Selective association of ion channels with specific lipid microenvironments within the membrane is thus likely to be an important and fundamental regulatory aspect of channel physiology. This brief review highlights some of the existing evidence for raft modulation of channel function.

ATP-sensitive potassium channels (K(ATP) channels) of arterial smooth muscle are important regulators of arterial tone, and hence blood flow, in response to vasoactive transmitters. Recent biochemical and electron microscopic evidence suggests that these channels localise to small vesicular invaginations of the plasma membrane, known as caveolae, and interact with the caveolae-associated protein, caveolin. Here we report that interaction with caveolin functionally regulates the activity of the vascular subtype of K(ATP) channel, Kir6.1/SUR2B. Pinacidil-evoked recombinant whole-cell Kir6.1/SUR2B currents recorded in HEK293 cells stably expressing caveolin-1 (69.6 +/- 8.3 pA pF(1), n = 8) were found to be significantly smaller than currents recorded in caveolin-null cells (179.7 +/- 35.9 pA pF(1), n = 6; P < 0.05) indicating that interaction with caveolin may inhibit channel activity. Inclusion in the pipette-filling solution of a peptide corresponding to the scaffolding domain of caveolin-1 had a similar inhibitory effect on whole-cell Kir6.1/SUR2B currents as co-expression with full-length caveolin-1, while a scrambled version of the same peptide had no effect. Interestingly, intracellular dialysis of vascular smooth muscle cells with the caveolin-1 scaffolding domain peptide (SDP) also caused inhibition of pinacidil-evoked native whole-cell K(ATP) currents, indicating that a significant proportion of vascular K(ATP) channels are susceptible to block by exogenously applied SDP. In cell-attached recordings of Kir6.1/SUR2B single channel activity, the presence of caveolin-1 significantly reduced channel open probability (from 0.05 +/- 0.01 to 0.005 +/- 0.001; P < 0.05) and the amount of time spent in a relatively long-lived open state. These changes in kinetic behaviour can be explained by a caveolin-induced shift in the channel's sensitivity to its physiological regulator MgADP. Our findings thus suggest that interaction with caveolin-1 suppresses vascular-type K(ATP) channel activity. Since caveolin expression is regulated by cellular free cholesterol and plasma levels of low-density lipoprotein (LDL), this interaction may have implications in both the physiological and pathophysiological control of vascular function.

Exchange proteins directly activated by cyclic AMP (Epacs or cAMP-GEF) represent a family of novel cAMP-binding effector proteins. The identification of Epacs and the recent development of pharmacological tools that discriminate between cAMP-mediated pathways have revealed previously unrecognized roles for cAMP that are independent of its traditional target cAMP-dependent protein kinase (PKA). Here we show that Epac exists in a complex with vascular ATP-sensitive potassium (KATP) channel subunits and that cAMP-mediated activation of Epac modulates KATP channel activity via a Ca2+-dependent mechanism involving the activation of Ca2+-sensitive protein phosphatase 2B (PP-2B, calcineurin). Application of the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP, at concentrations that activate Epac but not PKA, caused a 41.6 +/- 4.7% inhibition (mean +/- S.E.M.; n = 7) of pinacidil-evoked whole-cell KATP currents recorded in isolated rat aortic smooth muscle cells. Importantly, similar results were obtained when cAMP was elevated by addition of the adenylyl cyclase activator forskolin in the presence of the structurally distinct PKA inhibitors, Rp-cAMPS or KT5720. Activation of Epac by 8-pCPT-2'-O-Me-cAMP caused a transient 171.0 +/- 18.0 nM (n = 5) increase in intracellular Ca2+ in Fura-2-loaded aortic myocytes, which persisted in the absence of extracellular Ca2+. Inclusion of the Ca2+-specific chelator BAPTA in the pipette-filling solution or preincubation with the calcineurin inhibitors, cyclosporin A or ascomycin, significantly reduced the ability of 8-pCPT-2'-O-Me-cAMP to inhibit whole-cell KATP currents. These results highlight a previously undescribed cAMP-dependent regulatory mechanism that may be essential for understanding the physiological and pathophysiological roles ascribed to arterial KATP channels in the control of vascular tone and blood flow.

The traditional view of the plasma membrane as a uniform cellular envelope formed from a homogenous mixture of lipids has been refined in recent years to reflect the heterogeneity of its composite lipids. The membrane can consist of upwards of 500 different types of lipids, which exhibit complex and dynamic interactions. Cholesterol and sphingolipids, in particular, partition away from the bulk of bilayer to form distinct microdomains or 'rafts'. Although controversial, lipid rafts have attracted considerable attention over recent years, firstly because of their apparent ability to selectively aggregate interacting signalling molecules, including ion channels and receptors, and secondly because of the implication that they may be involved in the spatial organisation of signalling pathways. Here we describe methods to isolate lipid rafts, assess their purity and determine the distribution of potassium channel proteins between raft and non-raft fractions.

The functional localization of potassium inward rectifiers is regulated by SAP97, a PDZ membrane-associated guanylate kinase protein. We describe here an investigation of the conformation of the PDZ domain region of SAP97 PDZ1-3. The NMR and SAXS data reveal conformational dynamics. The NMR data show minimal interdomain contacts, with the U3 linker region between PDZ2 and PDZ3 being largely unstructured. Shape analysis of the SAXS profiles revealed a dumbbell for the PDZ12 double domain. An overall elongated, asymmetric shape comprised of two to three distinct components characterizes the triple domain PDZ1-3. In addition, rigid body modeling shows that the representative average shape does not provide the full picture and that the data for the triple domain are consistent with large variations, suggesting significant conformational flexibility. However, the dynamics appears to be restricted as PDZ3 is located essentially within approximately 40 A from PDZ12. We also show that the Kir2.1 cytoplasmic domain interacts with all three PDZ domains but with a clear preference for PDZ2 even in the presence of the U3 region. We speculate that the restricted dynamics and preferential Kir2.1 binding to PDZ2 are features that enable SAP97 to function as a scaffold protein, allowing other proteins each to bind to the other two PDZ domains in sufficient proximity to yield productive channelosomes.

Objective: The vasoconstrictor angiotensin II (Ang II) acts at G(q/11)-coupled receptors to suppress ATP-sensitive potassium (K(ATP)) channel activity via activation of protein kinase C (PKC). The aim of this study was to determine the PKC isoforms involved in the Ang II-induced inhibition of aortic K(ATP) channel activity and to investigate potential mechanisms by which these isoforms specifically target these ion channels., Methods and Results: We show that the inhibitory effect of Ang II on pinacidil-evoked whole-cell rat aortic K(ATP) currents persists in the presence of Gö6976, an inhibitor of the conventional PKC isoforms, but is abolished by intracellular dialysis of a selective PKCepsilon translocation inhibitor peptide. This suggests that PKC-dependent inhibition of aortic K(ATP) channels by Ang II arises exclusively from the activation and translocation of PKCepsilon. Using discontinuous sucrose density gradients and Western blot analysis, we show that Ang II induces the translocation of PKCepsilon to cholesterol-enriched rat aortic smooth muscle membrane fractions containing both caveolin, a protein found exclusively in caveolae, and Kir6.1, the pore-forming subunit of the vascular K(ATP) channel. Immunogold electron microscopy of rat aortic smooth muscle plasma membrane sheets confirms both the presence of Kir6.1 in morphologically identifiable regions of the membrane rich in caveolin and Ang II-evoked migration of PKCepsilon to these membrane compartments., Conclusions: Ang II induces the recruitment of the novel PKC isoform, PKCepsilon, to arterial smooth muscle caveolae. This translocation allows PKCepsilon access to K(ATP) channels compartmentalized within these specialized membrane microdomains and highlights a potential role for caveolae in targeting PKC isozymes to an ion channel effector.

The stress-activated protein kinase p38 and nitric oxide (NO) are proposed downstream effectors of excitotoxic cell death. Although the postsynaptic density protein PSD95 can recruit the calcium-dependent neuronal NO synthase (nNOS) to the mouth of the calcium-permeable NMDA receptor, and depletion of PSD95 inhibits excitotoxicity, the possibility that selective uncoupling of nNOS from PSD95 might be neuroprotective is unexplored. The relationship between excitotoxic stress-generated NO and activation of p38, and the significance of the PSD95-nNOS interaction to p38 activation also remain unclear. We find that NOS inhibitors reduce both glutamate-induced p38 activation and the resulting neuronal death, whereas NO donor has effects consistent with NO as an upstream regulator of p38 in glutamate-induced cell death. Experiments using a panel of decoy constructs targeting the PSD95-nNOS interaction suggest that this interaction and subsequent NO production are critical for glutamate-induced p38 activation and the ensuing cell death, and demonstrate that the PSD95-nNOS interface provides a genuine possibility for design of neuroprotective drugs with increased selectivity.

Arterial ATP-sensitive K+ (K(ATP)) channels are critical regulators of vascular tone, forming a focal point for signaling by many vasoactive transmitters that alter smooth muscle contractility and so blood flow. Clinically, these channels form the target of antianginal and antihypertensive drugs, and their genetic disruption leads to hypertension and sudden cardiac death through coronary vasospasm. However, whereas the biochemical basis of K(ATP) channel modulation is well-studied, little is known about the structural or spatial organization of the signaling pathways that converge on these channels. In this study, we use discontinuous sucrose density gradients and Western blot analysis to show that K(ATP) channels localize with an upstream signaling partner, adenylyl cyclase, to smooth muscle membrane fractions containing caveolin, a protein found exclusively in cholesterol and sphingolipid-enriched membrane invaginations known as caveolae. Furthermore, we show that an antibody against the K(ATP) pore-forming subunit, Kir6.1 co-immunoprecipitates caveolin from arterial homogenates, suggesting that Kir6.1 and caveolin exist together in a complex. To assess whether the colocalization of K(ATP) channels and adenylyl cyclase to smooth muscle caveolae has functional significance, we disrupt caveolae with the cholesterol-depleting agent, methyl-beta-cyclodextrin. This reduces the cAMP-dependent protein kinase A-sensitive component of whole-cell K(ATP) current, indicating that the integrity of caveolae is important for adenylyl cyclase-mediated channel modulation. These results suggest that to be susceptible to protein kinase A-dependent activation, arterial K(ATP) channels need to be localized in the same lipid compartment as adenylyl cyclase; the results also provide the first indication of the spatial organization of signaling pathways that regulate K(ATP) channel activity.

Inwardly rectifying potassium (Kir) channels are prime determinants of resting membrane potential in neurons. Their subcellular distribution and surface density thus help shape neuronal excitability, yet mechanisms governing the membrane targeting and localization of Kir channels are poorly understood. Here we report a direct interaction between the strong inward rectifier, Kir2.1, and a recently identified splice variant of postsynaptic density-93 (PSD-93), a protein involved the subcellular targeting of ion channels and glutamate receptors at excitatory synapses. Yeast two-hybrid screening of a human brain cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding the first two PDZ domains of PSD-93, but with an extended N-terminal region that diverged from other PSD-93 isoforms. This clone represented the human homologue of the mouse PSD-93 splice variant, PSD-93delta. Reverse transcription-polymerase chain reaction analysis showed diffuse low level PSD-93delta expression throughout the brain, with significantly higher levels in spinal cord. In vitro binding studies revealed that a type I PDZ recognition motif at the extreme C terminus of the Kir2.1 mediates interaction with all three PDZ domains of PSD-93delta, and association between Kir2 channels and PSD-93delta was confirmed further by the ability of anti-Kir2.1 antibodies to coimmunoprecipitate PSD-93delta from rat spinal cord lysates. Functionally, coexpression of Kir2.1 and PSD-93delta had no discernible effect upon channel kinetics but resulted in cell surface Kir2.1 clustering and suppression of channel internalization. We conclude that PSD-93delta is potentially an important regulator of the spatial and temporal distribution of Kir2 channels within neuronal membranes of the central nervous system.

The role of filamins in actin cross-linking and membrane stabilization is well established, but recently their ability to interact with a variety of transmembrane receptors and signaling proteins has led to speculation of additional roles in scaffolding and signal transduction. Here we report a direct interaction between filamin-A and Kir2.1, an isoform of inwardly rectifying potassium channel expressed in vascular smooth muscle and an important regulator of vascular tone. Yeast two-hybrid screening of a porcine coronary artery cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding a fragment of filamin-A (residues 2481-2647). Interaction between filamin-A and Kir2.1 was confirmed by in vitro overlay assay of membrane-bound Kir2.1 with glutathione S-transferase fusion protein of the isolated filamin clone. Additionally, antibodies directed against Kir2.1 coimmunoprecipitated filamin-A from arterial smooth muscle cell lysates, and immunocytochemical analysis of individual arterial smooth muscle cells showed that Kir2.1 and filamin co-localize in "hotspots" at the cell membrane. Interaction with filamin-A was found to have no effect on Kir2.1 channel behavior but, rather, increased the number of functional channels resident within the membrane. We conclude that filamin-A is potentially an important regulator of Kir2.1 surface expression and location within vascular smooth muscle.