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Lysosomes have fundamental physiological roles and have previously been implicated in Parkinson's disease.sup.1-5. However, how extracellular growth factors communicate with intracellular organelles to control lysosomal function is not well understood. Here we report a lysosomal K.sup.+ channel complex that is activated by growth factors and gated by protein kinase B (AKT) that we term lysoK.sub.GF. LysoK.sub.GF consists of a pore-forming protein TMEM175 and AKT: TMEM175 is opened by conformational changes in, but not the catalytic activity of, AKT. The minor allele at rs34311866, a common variant in TMEM175, is associated with an increased risk of developing Parkinson's disease and reduces channel currents. Reduction in lysoK.sub.GF function predisposes neurons to stress-induced damage and accelerates the accumulation of pathological [alpha]-synuclein. By contrast, the minor allele at rs3488217--another common variant of TMEM175, which is associated with a decreased risk of developing Parkinson's disease--produces a gain-of-function in lysoK.sub.GF during cell starvation, and enables neuronal resistance to damage. Deficiency in TMEM175 leads to a loss of dopaminergic neurons and impairment in motor function in mice, and a TMEM175 loss-of-function variant is nominally associated with accelerated rates of cognitive and motor decline in humans with Parkinson's disease. Together, our studies uncover a pathway by which extracellular growth factors regulate intracellular organelle function, and establish a targetable mechanism by which common variants of TMEM175 confer risk for Parkinson's disease. The identification of a lysosomal K.sup.+ channel complex--comprising AKT and the pore-forming TMEM175--provides insights into the mechanisms through which variants of the pore-forming protein affect the development of Parkinson's disease., Author(s): Jinhong Wie [sup.1] , Zhenjiang Liu [sup.1] , Haikun Song [sup.2] , Thomas F. Tropea [sup.3] , Lu Yang [sup.4] , Huanhuan Wang [sup.4] , Yuling Liang [sup.5] , [...]

The essential but enigmatic functions of sleep.sup.1,2 must be reflected in molecular changes sensed by the brain's sleep-control systems. In the fruitfly Drosophila, about two dozen sleep-inducing neurons.sup.3 with projections to the dorsal fan-shaped body (dFB) adjust their electrical output to sleep need.sup.4, via the antagonistic regulation of two potassium conductances: the leak channel Sandman imposes silence during waking, whereas increased A-type currents through Shaker support tonic firing during sleep.sup.5. Here we show that oxidative byproducts of mitochondrial electron transport.sup.6,7 regulate the activity of dFB neurons through a nicotinamide adenine dinucleotide phosphate (NADPH) cofactor bound to the oxidoreductase domain.sup.8,9 of Shaker's K.sub.V[beta] subunit, Hyperkinetic.sup.10,11. Sleep loss elevates mitochondrial reactive oxygen species in dFB neurons, which register this rise by converting Hyperkinetic to the NADP.sup.+-bound form. The oxidation of the cofactor slows the inactivation of the A-type current and boosts the frequency of action potentials, thereby promoting sleep. Energy metabolism, oxidative stress, and sleep--three processes implicated independently in lifespan, ageing, and degenerative disease.sup.6,12-14--are thus mechanistically connected. K.sub.V[beta] substrates.sup.8,15,16 or inhibitors that alter the ratio of bound NADPH to NADP.sup.+ (and hence the record of sleep debt or waking time) represent prototypes of potential sleep-regulatory drugs. Sleep deprivation in Drosophila elevates reactive oxygen species in sleep-promoting neurons, leading to changes in potassium currents and spiking activity and thereby connecting energy metabolism, oxidative stress, and sleep., Author(s): Anissa Kempf [sup.1] , Seoho M. Song [sup.1] , Clifford B. Talbot [sup.1] , Gero Miesenböck [sup.1] Author Affiliations: (1) Centre for Neural Circuits and Behaviour, University of Oxford, [...]

The purpose of this study was to determine the relationship of [K.sub.Ca] channels to endothelium-dependent hyperpolarizing factor (EDHF)-mediated relaxation induced by acetylcholine (ACh) in the superior mesenteric arteries of 7-month- old spontaneously hypertensive rats (SHR). Upon inhibition of nitric oxide synthase and cyclooxygenase, ACh-induced EDHF-mediated relaxation was found to be weaker in SHR than in age-matched Wistar Kyoto rats (WKY). These relaxations in both group were attenuated by combined treatment with small-conductance and intermediate-conductance [Ca.sub.2+]-activated [K.sup.+] channels ([SK.sub.Ca] and [IK.sub.Ca]) inhibitors, with the exception of relaxation resistant to inhibition of these channels in SHR (vs. WKY). Treatment with large-conductance [Ca.sup.2+]-activated [K.sup.+] channels ([BK.sub.Ca]) inhibitor specifically attenuated relaxation in SHR, but not in WKY. Protein expression of [IK.sub.Ca] and [SK.sub.Ca] in the arteries did not differ between the 2 groups, whereas ratio of slo[beta]1 subunit to a subunit of [BK.sub.Ca] was increased in SHR (vs. WKY). These results suggest that EDHF-mediated relaxations in superior mesenteric arteries are impaired in SHR, and utilize components of [BK.sub.Ca] in addition to [SK.sub.Ca]/[IK.sub.Ca] channel activities, that the increased participation of [BK.sub.Ca] may be attributable to alterations in a and slo[beta]1 subunit ratio, and that components unrelated to [K.sub.Ca] activity may also contribute to the difference between SHR and WKY arteries. Key words: calcium-activated potassium channel, endothelium-dependent hyperpolarization, hypertension, superior mesenteric artery, SHR. Cette etude avait pour but d'etablir le lien entre les canaux [K.sub.Ca] et la relaxation produite par l'acetylcholine (ACh) dans les arteres mesenteriques superieures de rats spontanement hypertendus (SHR) de 7 mois, et mediee par le facteur hyperpolarisant dependant de l'endothelium (EDHF). Nous avons observe que dans l'inhibition de l'oxyde nitrique synthase et de la cyclooxygenase, la relaxation produite par l'acetylcholine et mediee par l'EDHF etait moins importante chez les rats SHR que chez des rats Wistar Kyoto (WKY) apparies pour l'age. Ces relaxations observees dans les deux groupes s'attenuaient avec l'administration combinee d'inhibiteurs des canaux [K.sup.+] actives par le [Ca.sup.2+] de faible (<>) conductance ([SK.sub.Ca]) et de conductance intermediate ([IK.sub.Ca]), a l'exception d'une relaxation plus resistante a l'inhibition de ces canaux chez les rats SHR que chez les rats WKY. L'administration d'un inhibiteur des canaux [K.sup.+] actives par le [Ca.sup.2+] de haute (<>) conductance ([BK.sub.Ca]) entramait une attenuation specifique de la relaxation chez les rats SHR, mais pas chez les rats WKY. Nous n'avons observe dans les arteres aucune difference dans l'expression proteique des canaux [IK.sub.Ca] et [SK.sub.Ca] entre les deux groupes, tandis que le ratio entre les sous-unites slo[beta]1 et a des canaux [BK.sub.Ca] augmentait davantage chez les rats SHR que chez les rats WKY. Ces resultats laissent entendre (i) que les relaxations mediees par l'EDHF dans les arteres mesenteriques superieures seraient diminuees chez les rats SHR et qu'elle utiliseraient une composante de canaux [BK.sub.Ca] en plus de l'activite des canaux [SK.sub.Ca]/[IK.sub.Ca], (ii) que l'augmentation de la participation des canaux [BK.sub.Ca] pourrait etre attribuable a des modifications du ratio entre les sous-unites a et slopi, et (iii) que des composantes etrangeres a l'activite des canaux KCa pourrait aussi contribuer aux differences entre les arteres de rats SHR et de rats WKY. [Traduit par la Redaction] Mots-cles: canal potassique active par le calcium, hyperpolarisation dependante de l'endothelium, hypertension, artere mesenterique superieure, rat spontanement hypertendu., Introduction The endothelium plays a crucial role in the regulation of vascular tone and therefore endothelial dysfunction is a contributing factor in hypertension and hypertension-associated vascular complications (Matsumoto et al. [...]

In the wake of demographic change in Western countries, atrial fibrillation has reached an epidemiological scale, yet current strategies for drug treatment of the arrhythmia lack sufficient efficacy and safety. In search of novel medications, atrial-selective drugs that specifically target atrial over other cardiac functions have been developed. Here, I will address drugs acting on potassium ([K.sup.+]) channels that are either predominantly expressed in atria or possess electrophysiological properties distinct in atria from ventricles. These channels include the ultra-rapidly activating, delayed outward-rectifying Kv1.5 channel conducting [I.sub.Kur], the acetylcholine-activated inward-rectifying Kir3.1/Kir3.4 channel conducting [I.sub.K,Ach], the [Ca.sup.2+]-activated [K.sup.+] channels of small conductance (SK) conducting [I.sub.SK], and the two-pore domain [K.sup.+] (K2P) channels (tandem of P domains, weak inward-rectifying [K.sup.+] channels (TWIK-1), TWIK-related acid-sensitive [K.sup.+] channels (TASK-1 and TASK-3)) that are responsible for voltage-independent background currents [I.sub.TWIK-1], [I.sub.TASK-1], and [I.sub.TASK-3]. Direct drug effects on these channels are described and their putative value in treatment of atrial fibrillation is discussed. Although many potential drug targets have emerged in the process of unravelling details of the pathophysiological mechanisms responsible for atrial fibrillation, we do not know whether novel antiarrhythmic drugs will be more successful when modulating many targets or a single specific one. The answer to this riddle can only be solved in a clinical context. Key words: atrial fibrillation, antiarrhythmic drugs, voltage-dependent [K.sup.+] channels, two-pore domain [K.sup.+] channels, [Ca.sup.2+]- activated [K.sup.+] channels. Resume: En raison de changements demographiques dans les pays occidentaux, la fibrillation auriculaire a atteint des proportions epidemiques, mais les strategies actuelles du traitement medicamenteux de l'arythmie sont insuffisantes en matiere d'efficacite et d'innocuite. A la recherche de nouveaux medicaments, on a developpe des medicaments selectifs pour les oreillettes qui ciblent l'oreillette dans une plus grande mesure que d'autres fonctions cardiaques. Dans la presente synthese, je me pencherai sur des medicaments agissant au niveau des canaux potassiques ([K.sup.+]) qui sont soit exprimes de maniere predominante dans les oreillettes, soit dotes de proprietes electrophysiologiques distinctes dans les oreillettes par rapport aux ventricules. Ces canaux comprennent le canal Kv1.5 (a rectification retardee sortante activee ultra-rapidement) conduisant le courant [I.sub.Kur], le canal Kir3.1/Kir3.4 (a rectification entrante activee par l'acetylcholine) conduisant le courant [I.sub.K,ACh], le canal SK ([K.sup.+] de faible conductance active par le [Ca.sup.2+]) conduisant le courant [I.sub.SK] et les canaux K2P ([K.sup.+] a deux domaines pore avec un couple de domaines P de canaux TWIK-1 ([K.sup.+] a rectification entrante) ainsi que de canaux TASK-1 et TASK-3 ([K.sup.+] lies a TWIK sensibles a l'acidification)) qui sont responsables des courants de fond [I.sub.TWIK-1'] [I.sub.TASK-1] et [I.sub.TASK- 3] (independants du voltage). Je decris les effets directs des medicaments sur ces canaux, et je discute de leur valeur presumee dans le traitement de la fibrillation auriculaire. Bien que de nombreuses cibles medicamenteuses potentielles aient emerge au cours de l'eclaircissement des modes d'action physio-pathologiques responsables de la fibrillation auriculaire, nous ne savons pas si de nouveaux antiarythmiques auront plus de succes s'ils participent a la modulation de nombreuses cibles ou d'une seule cible specifique. Il s'agit d'une enigme qui peut resoudre uniquement dans un contexte clinique. [Traduit par la Redaction] Mots-cles: fibrillation auriculaire, antiarythmiques, canaux [K.sup.+] dependants du voltage, canaux [K.sup.+] a deux domaines pore, canaux [K.sup.+] actives par l'ion [Ca.sup.2+]., Introduction Atrial fibrillation (AF) affects more than 6 million Europeans (European Heart Rhythm Association et al. 2010), worsening their quality of life and increasing their mortality due to elevated risk [...]

Gabapentin as an anticonvulsant drug also has beneficial effects in treatment of depression. Previously, we showed that acute administration of gabapentin produced an antidepressant-like effect in the mouse forced swimming test (FST) by a mechanism that involves the inhibition of nitric oxide (NO). Considering the involvement of NO in adenosine triphosphate (ATP)-sensitive potassium channels ([K.sub.ATP]), in the present study we investigated the involvement of [K.sub.ATP] channels in antidepressant-like effect of gabapentin. Gabapentin at different doses (5-10 mg/kg) and fluoxetine (20 mg/kg) were administrated by intraperitoneal route, 60 and 30 min, respectively, before the test. To clarify the probable involvement of [K.sub.ATP] channels, mice were pretreated with [K.sub.ATP] channel inhibitor or opener. Gabapentin at dose 10 mg/kg significantly decreased the immobility behavior of mice similar to fluoxetine (20 mg/kg). Co-administration of subeffective dose (1 mg/kg) of glibenclamide (inhibitor of [K.sub.ATP] channels) with gabapentin (3 mg/kg) showed a synergistic antidepressant-like effect. Also, subeffective dose of cromakalim (opener of [K.sub.ATP] channels, 0.1 mg/kg) inhibited the antidepressant-like effect of gabapentin (10 mg/kg). None of the treatments had any impact on the locomotor movement. Our study, for the first time, revealed that antidepressant-like effect of gabapentin in mice is mediated by blocking the [K.sub.ATP] channels. Key words: gabapentin, ATP-sensitive potassium channels ([K.sub.ATP]), forced swimming test, mice. La gabapentine est un anticonvulsivant qui permet aussi d'obtenir des bienfaits dans le traitement de la depression. Nous avons deja montre que l'administration aigue de gabapentine produit un effet s'apparentant a celui des antidepresseurs dans le test de nage forcee chez la souris, par un mode d'action qui met en jeu l'inhibition des effets de l'oxyde nitrique (NO). Etant donne la participation du NO au fonctionnement des canaux potassiques sensibles a l'adenosine triphosphate ([K.sub.ATP]), dans les presents travaux, nous avons etudie la participation des canaux [K.sub.ATP] dans les effets de la gabapentine s'apparentant a ceux des antidepresseurs. Nous avons administre par voie intraperitoneale differentes doses de gabapentine (5-10 mg/kg) et de la fluoxetine (20 mg/kg), respectivement 60 et 30 min avant le test. Afin de clarifier le role probable des canaux [K.sub.ATP], nous avons expose au prealable les souris a un inhibiteur ou a un activateur des canaux [K.sub.ATP]. La gabapentine a 10 mg/kg a permis de diminuer le comportement d'immobilite des souris dans une mesure equivalente a la fluoxetine (20 mg/kg). L'administration conjointe de glibenclamide (un inhibiteur des canaux [K.sub.ATP]) a des doses inferieures aux doses efficaces (1 mg/kg) et de gabapentine (3 mg/kg) a revele la presence d'un effet synergique s'apparentant a ceux des antidepresseurs. De plus, l'administration de cromakalim (un activateur des canaux [K.sub.ATP]) a des doses inferieures aux doses efficaces (0,1 mg/kg) a entraine une inhibition des effets de la gabapentine (10 mg/kg) s'apparentant a ceux des antidepresseurs. Aucun des medicaments n'avait d'incidence sur les mouvements de locomotion. Notre etude est la premiere a montrer que l'inhibition des canaux [K.sub.ATP] assure la mediation des effets de la gabapentine s'apparentant a ceux des antidepresseurs chez la souris. [Traduit par la Redaction] Mots-cles: gabapentine, canaux potassiques sensibles a l'ATP ([K.sub.ATP]), test de nage forcee, souris., Introduction Depression is one of the serious mood disorders. Due to its high frequency of occurrence, it acts as a main psychiatric problems in public health concerns (Silva et al. [...]

The blood-brain barrier (BBB) is an integral part of the neurovascular unit (NVU). The NVU is comprised of endothelial cells that are interconnected by tight junctions resting on a parenchymal [...]

High-conductance voltage- and [Ca.sup.2+]-activated [K.sup.+] channels function in many physiological processes that link cell membrane voltage and intracellular [Ca.sup.2+] concentration, including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning (1-8). Like other voltage-dependent [K.sup.+] channels, [Ca.sup.2+]-activated [K.sup.+] channels open when the cell membrane depolarizes, but in contrast to other voltage-dependent [K.sup.+] channels, they also open when intracellular [Ca.sup.2+] concentrations rise. Channel opening by [Ca.sup.2+] is made possible by a structure called the gating ring, which is located in the cytoplasm. Recent structural studies have defined the [Ca.sup.2+]-free, closed, conformation of the gating ring, but the [Ca.sup.2+]-bound, open, conformation is not yet known (9). Here we present the [Ca.sup.2+]-bound conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of [Ca.sup.2+], opens like the petals of a flower. The degree to which it opens explains how [Ca.sup.2+] binding can open the transmembrane pore. These findings present a molecular basis for [Ca.sup.2+] activation of [K.sup.+] channels and suggest new possibilities for targeting the gating ring to treat conditions such as asthma and hypertension., Regulators of [K.sup.+] conductance (RCK) domains are ubiquitous among ion channels and transporters in prokaryotic cells (10-14). Eight RCK domains assemble in the cytoplasm to form a closed-ring structure that [...]

The renal outer medullary [K.sup.+] (ROMK) channel plays a critical role in renal sodium handling. Recent genome sequencing efforts in the Framingham Heart Study offspring cohort (Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, and Lifton RP. Nat Genet 40: 592-599, 2008) recently revealed an association between suspected loss-of-function polymorphisms in the ROMK channel and resistance to hypertension, suggesting that ROMK activity may also be a determinant of blood pressure control in the general population. Here we examine whether these sequence variants do, in fact, alter ROMK channel function and explore the mechanisms. As assessed by two-microelectrode voltage clamp in Xenopus oocytes, 3/5 of the variants (R193P, H251Y, and T313FS) displayed an almost complete attenuation of whole cell ROMK channel activity. Surface antibody binding measurements of external epitope-tagged channels and analysis of glycosylation-state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of retention in the endoplasmic reticulum. The other variants (P166S, Rl69H) had no obvious effects on the basal channel activity or surface expression but, instead, conferred a gain in regulated-inhibitory gating. As assessed in giant excised patch-clamp studies, apparent phosphotidylinositol 4,5-bisphosphate (PIP2) binding affinity of the variants was reduced, causing channels to be more susceptible to inhibition upon [PIP.sub.2] depletion. Unlike the protein product of the major ROMK allele, these two variants are sensitive to the inhibitory affects of a G proteincoupled receptor, which stimulates [PIP.sub.2] hydrolysis. In summary, we have found that hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in the maintenance of blood pressure. inward rectifier; potassium channel; blood pressure; inward rectified potassium channel 1.1; Bartter; phosphotidylinositol 4,5-bisphosphate; misfolded protein doi: 10.1152/ajprenal.000257.2010

The KCNQI [K.sup.+] channel plays a key role in the regulation of several physiological functions, including cardiac excitability, cardiovascular tone, and body electrolyte homeostasis. The metabolic sensor AMP-activated protein kinase (AMPK) has been shown to regulate a growing number of ion transport proteins. To determine whether AMPK regulates KCNQ1, we studied the effects of AMPK activation on KCNQ1 currents in Xenopus laevis oocytes and collecting duct epithelial cells. AMPK activation decreased KCNQ1 currents and channel surface expression in X. laevis oocytes, but AMPK did not phosphorylate KCNQI in vitro, suggesting an indirect regulatory mechanism. As it has been recently shown that the ubiquitin-protein ligase Nedd4-2 inhibits KCNQI plasma membrane expression and that AMPK regulates epithelial [Na.sup.+] channels via Nedd4-2, we examined the role of Nedd4-2 in the AMPK-dependent regulation of KCNQl. Channel inhibition by AMPK was blocked in oocytes coexpressing either a dominant-negative or constitutively active Nedd4-2 mutant, or a Nedd4-2 interaction-deficient KCNQ1 mutant, suggesting that Nedd4-2 participates in the regulation of KCNQ1 by AMPK. KCNQ1 is expressed at the basolateral membrane in mouse polarized kidney cortical collecting duct ([mpkCCD.sub.c14]) cells and in rat kidney. Treatment with the AMPK activators AICAR (2 mM) or metformin (1 mM) reduced basolateral KCNQ1 currents in apically permeabilized polarized [mpkCCD.sub.c14] cells. Moreover, AICAR treatment of rat kidney slices ex vivo induced AMPK activation and intracellular redistribution of KCNQ1 from the basolateral membrane in collecting duct principal cells. AICAR treatment also induced increased ubiquitination of KCNQ1 immunoprecipitated from kidney slice homogenates. These results indicate that AMPK inhibits KCNQ 1 activity by promoting Nedd4-2-dependent channel ubiquitination and retrieval from the plasma membrane. KCNEI; ion channels; potassium transport doi: 10.1152/ajprenal.00423.2010

The voltage-gated potassium channel Kvl.3 has been recently identified as a molecular target that allows the selective pharmacological suppression of effector memory T cells ([T.sub.EM]) without affecting the function of naive T cells (TN) and central memory T cells ([T.sub.CM]). We found that Kvl.3 was expressed on glomeruli and some tubules in rats with anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). A flow cytometry analysis using kidney cells revealed that most of the [CD4.sup.+] T cells and some of the [CD8.sup.+] T cells had the [T.sub.EM] phenotype ([CD45RC.sup.-][CD62L.sup.-]). Double immunofluorescence staining using mononuclear cell suspensions isolated from anti-GBM GN kidney showed that Kvl.3 was expressed on T cells and some macrophages. We therefore investigated whether the Kvl.3 blocker Psora-4 can be used to treat anti-GBM GN. Rats that had been given an injection of rabbit anti-rat GBM antibody were also injected with Psora-4 or the vehicle intraperitoneally. Rats given Psora-4 showed less proteinuria and fewer crescentic glomeruli than rats given the vehicle. These results suggest that [T.sub.EM] and some macrophages expressing Kvl.3 channels play a critical role in the pathogenesis of crescentic GN and that Psora-4 will be useful for the treatment of rapidly progressive glomerulonephritis. WKY rats; crescentic glomerulonephritis; flow cytometric analysis doi: 10.1152/ajprenal.00374.2010

Multiple, perhaps interactive, mechanisms participate in the linkage between increased neural activity and cerebral vasodilation. In the present study, we assessed whether neural activation-related pial arteriolar dilation (PAD) involved interactions among adenosine (Ado) [A.sub.2] receptors ([A.sub.2]Rs), large-conductance [Ca.sup.2+]-operated [K.sup.+] ([BK.sub.Ca]) channels, and inward rectifier [K.sup.+] ([K.sub.ir]) channels. In rats with closed cranial windows, we monitored sciatic nerve stimulation (SNS)-induced PAD in the absence or presence of pharmacological blockade of [A.sub.2]Rs (ZM-241385), ecto-5'-nucleotidase ([alpha],[beta]-methylene-adenosine diphosphate), [BK.sub.Ca] channels (paxilline), and [K.sub.ir] channels (Ba[Cl.sub.2]). Individually, these interventions led to 53-66% reductions in SNS-induced PADs. Combined applications of these blockers led to little or no further repression of SNS-induced PADs, suggesting interactions among [A.sub.2]Rs and [K.sup.+] channels. In the absence of SNS, Ba[Cl.sub.2] blockade of [K.sub.ir] channels produced 52-80% reductions in Ado and NS-1619 ([BK.sub.Ca] channel activator)-induced PADs. In contrast, paxilline blockade of [BK.sub.Ca] channels was without effect on dilations elicited by KCl ([K.sub.ir] channel activator) and Ado suffusions, indicating that Ado- and NS-1619-associated PADs involved [K.sub.ir] channels. In addition, targeted ablation of the superficial glia limitans was associated with a selective 60-80% loss of NS-1619 responses, suggesting that the [BK.sub.Ca] channel participation (and paxilline sensitivity) derived largely from channels within the glia limitans. Additionally, blockade of either PKA or adenylyl cyclase caused markedly attenuated pial arteriolar responses to SNS and, in the absence of SNS, responses to Ado, KCl, and NS-1619. These findings suggested a key, possibly permissive, role for [A.sub.2]R-linked cAMP generation and PKA-induced [K.sup.+] channel phosphorylation in somatosensory activation-evoked PAD. neurovascular coupling; phosphorylation; adenylyl cyclase; cAMP-dependent protein kinase; cGMP-dependent protein kinase doi: 10.1152/ajpheart.00702.2010.

The sulfonylurea receptor-2 (SUR2) is a subunit of ATP-sensitive potassium channels (KATP) in heart. Mice with the SUR2 gene disrupted (SUR2m) are constitutively protected from ischemia-reperfusion (I/R) cardiac injury. This was surprising because [K.sub.ATP], either sarcolemmal or mitochondrial or both, are thought to be important for cardioprotection. We hypothesized that SUR2m mice have an altered mitochondrial phenotype that protects against I/R. Mitochondrial membrane potential ([DELTA][[PSI].sub.m]), tolerance to [Ca.sup.2+] load, and reactive oxygen species (ROS) generation were studied by fluorescence-based assays, and volumetric changes in response to [K.sup.+] were measured by light scattering in isolated mitochondria. For resting SUR2m mitochondria compared with wild type, the [DELTA][[PSI].sub.m] was less polarized (46.1 [+ or -] 0.4 vs. 51.9 [+ or -] 0.6%), tolerance to [Ca.sup.2+] loading was increased (163 [+ or -] 2 vs. 116 [+ or -] 2 [micro]M), and ROS generation was enhanced with complex I [8.5 [+ or -] 1.2 vs. 4.9 [+ or -] 0.2 arbitrary fluorescence units (afu)/s] or complex II (351 [+ or -] 51.3 vs. 166 [+ or -] 36.2 afu/s) substrates. SUR2m mitochondria had greater swelling in [K.sup.+] medium (30.2 [+ or -] 3.1%) compared with wild type (14.5 [+ or -] 0.6%), indicating greater [K.sup.+] influx. Additionally, [DELTA][[PSI].sub.m] decreased and swelling increased in the absence of ATP in SUR2m, but the sensitivity to ATP was less compared with wild type. When the mitochondria were subjected to hypoxia-reoxygenation, the decrease in respiration rates and respiratory control index was less in SUR2m. [DELTA][[PSI].sub.m] maintenance in the SUR2m intact myocytes was also more tolerant to metabolic inhibition. In conclusion, the cardioprotection observed in the SUR2m mice is associated with a protected mitochondrial phenotype resulting from enhanced K+ conductance that partially dissipated [DELTA][[PSI].sub.m]. These results have implications for possible SUR2 participation in mitochondrial [K.sub.ATP]. adenosine 5-triphosphate-sensitive potassium channel; sulfonylurea receptor 2; ischemia; ischemic-preconditioning; mitochondria; mitochondrial membrane potential; mitochondrial respiration; cellular energetics doi: 10.1152/ajpheart.00363.2010.

Small conductance [Ca.sup.2+]-activated [K.sup.+] channels (SK) regulate action potential (AP) firing properties and excitability in many central neurons. However, the functional roles of SK channels of parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal days 7-9. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. With the use of whole cell current clamp, single APs and spike trains of different frequencies were evoked by current injections. We found that 1) PCMNs have two different firing patterns: the majority of PCMNs (90%) exhibited spike frequency adaptation (SFA) and the rest (10%) showed less or no adaptation; 2) application of the specific SK channel blocker apamin significantly increased spike half-width in single APs and trains and reduced the spike frequency-dependent AP broadening in trains; 3) SK channel blockade suppressed afterhyperpolarization (AHP) amplitude following single APs and trains and abolished spike-frequency dependence of AHP in trains; and 4) SK channel blockade increased the spike frequency but did not alter the pattern of SFA. Using whole cell voltage clamp, we measured outward currents and afterhyperpolarization current ([I.sub.AHP]). SK channel blockade revealed that SK-mediated outward currents had both transient and persistent components. After bath application of apamin and [Ca.sup.2+]-free solution, we found that apamin-sensitive and [Ca.sup.2+]-sensitive [I.sub.AHP] were comparable, confirming that SK channels may contribute to a major portion of [Ca.sup.2+]-activated [K.sup.+] channel-mediated [I.sub.AHP]. These results suggest that PCMNs have SK channels that significantly regulate AP repolarization, AHP, and spike frequency but do not affect SFA. We conclude that activation of SK channels underlies one of the mechanisms for negative control of PCMN excitability. repolarization; afterhyperpolarization; transient outward currents; afterhyperpolarization currents doi: 10.1152/ajpcell.00134.2010.

Gram negative pathogens are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. We have elucidated the mechanism of gating through structural and functional analysis of Escherichia coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that sense both reduced glutathione, which inhibits Kef activity, and glutathione adducts that form during electrophile detoxification and activate Kef. We find that reduced glutathione stabilizes an interdomain association between two KTN folds, whereas large adducts sterically disrupt this interaction. F441 is identified as the pivotal residue discriminating between reduced glutathione and its conjugates. We demonstrate a major structural change on the binding of an activating ligand to a KTN-domain protein. Analysis of the regulatory interactions suggests strategies to disrupt pathogen potassium and pH homeostasis. channel regulation | KTN structure doi/ 10.1073/pnas.1012716107

BK-type [K.sup.+] channels are activated by voltage and intracellular [Ca.sup.2+], which is important in modulating muscle contraction, neural transmission, and circadian pacemaker output. Previous studies suggest that the cytosolic domain of BK channels contains two different [Ca.sup.2+] binding sites, but the molecular composition of one of the sites is not completely known. Here we report, by systematic mutagenesis studies, the identification of E535 as part of this [Ca.sup.2+] binding site. This site is specific for binding to [Ca.sup.2+] but not [Cd.sup.2+]. Experimental results and molecular modeling based on the X-ray crystallographic structures of the BK channel cytosolic domain suggest that the binding of [Ca.sup.2+] by the side chains of E535 and the previously identified D367 changes the conformation around the binding site and turns the side chain of M513 into a hydrophobic core, providing a basis to understand how [Ca.sup.2+] binding at this site opens the activation gate of the channel that is remotely located in the membrane. [Ca.sup.2+]-activated | allosteric gating | [Ca.sup.2+] binding site | [Cd.sup.2+] | Slo1 doi/ 10.1073/pnas.1010124107

Granulocyte colony-stimulating factor (G-CSF) has been reported to improve cardiac function after myocardial infarction. However, whether postinfarct acute effect of G-CSF is mediated through the same signaling pathways as those of ischemic postconditioning is still unclear. We examined the postinfarct acute effect of G-CSF on myocardial infarct size and its precise molecular mechanism. Japanese white rabbits underwent 30 min of ischemia and 48 h of reperfusion. Rabbits were intravenously injected 10 [micro]g/kg of G-CSF (G-CSF group) or saline (control group) immediately after reperfusion. The wortmannin + G-CSF, PD-98059 + G-CSF, [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) + G-CSF, and 5-hydroxydecanoic acid sodium salt (5-HD) + G-CSF groups were respectively injected with wortmannin (0.6 mg/kg), PD-98059 (0.3 mg/kg), L-NAME (10 mg/kg), and 5-HD (5 mg/kg) 5 min before G-CSF administration. Myocardial infarct size was calculated as a percentage of the risk area of the left ventricle. Western blot analysis was performed to examine the signals such as protein kinase B (Akt), extracellular signal-regulated protein kinase (ERK), eNOS, p70S6 kinase (p70S6K), and glycogen synthase kinase-3[beta] (GSK3[beta]) in the ischemic myocardium after 48 h of reperfusion. The infarct size was significantly smaller in the G-CSF group (26.7 [+ or -] 2.7%) than in the control group (42.3 [+ or -] 4.6%). The infarct size-reducing effect of G-CSF was completely blocked by wortmannin (44.7 [+ or -] 4.8%), PD-98059 (38.3 [+ or -] 3.9%), L-NAME (42.1 [+ or -] 4.2%), and 5-HD (42.5 [+ or -] 1.7%). Wortmannin, PD-98059, L-NAME, or 5-HD alone did not affect the infarct size. Western blotting showed higher myocardial expression of phospho-Akt, phospho-ERK, phosho-eNOS, phosho-p70S6K, and phosho-GSK3[beta] at 10 min and 48 h after reperfusion in the G-CSF group than in the control group. In conclusion, postreperfusion G-CSF administration reduces myocardial infarct size via activation of phosphatidylinositol 3-kinase-Akt and ERK prosurvival signaling pathways and their downstream targets eNOS, p70S6 kinase, GSK3[beta], and mitochondrial ATP-dependent [K.sup.+] channel. granulocyte colony-stimulating factor; myocardial infarction; infarct size; protein kinase B; extracellular signal-regulated protein kinase; endothelial nitric oxide synthase; p70S6 kinase; glycogen synthase kinase-3[beta]; adenosine 5'-diphosphate-dependent potassium ion channels; reperfusion injury salvage kinase doi: 10.1152/ajpheart.00116.2010.

Sulfonylurea receptor-containing ATP-sensitive potassium ([K.sub.ATP]) channels have been implicated in cardioprotection, but the cell type and constitution of channels responsible for this protection have not been clear. Mice deleted for the first nucleotide binding region of sulfonylurea receptor 2 (SUR2) are referred to as SUR2 null since they lack full-length SUR2 and glibenclamide-responsive [K.sub.ATP] channels in cardiac, skeletal, and smooth muscle. As previously reported, SUR2 null mice develop electrocardiographic changes of ST segment elevation that were shown to correlate with coronary artery vasospasm. Here we restored expression of the cardiomyocyte SUR2-[K.sub.ATP] channel in SUR2 null mice by generating transgenic mice with ventricular cardiomyocyte-restricted expression of SUR2A. Introduction of the cardiomyocyte SUR2A transgene into the SUR2 null background restored functional cardiac [K.sub.ATP] channels. Hearts isolated from rescued mice, referred to as MLC2A, had significantly reduced infarct size (27 [+ or -] 3% of area at risk) compared with SUR2 null mice (36 [+ or -] 3% of area at risk). Compared with SUR2 null hearts, MLC2A hearts exhibited significantly improved cardiac function during the postischemia reperfusion period primarily because of preservation of low diastolic pressures. Additionally, restoration of cardiac SUR2-[K.sub.ATP] channels significantly reduced the degree and frequency of ST segment elevation episodes in MLC2A mice. Therefore, cardioprotective mechanisms both dependent and independent of SUR2-[K.sub.ATP] channels contribute to cardiac function. ATP-sensitive potassium channel; SUR2; SUR2A; ischemia; vasospasm doi: 10.1152/ajpheart.00084.2010.

Mechanosensitive large-conductance [Ca.sup.2+] or -activated [K.sup.+] channels encoded by the Slo1 gene ([BK.sub.Ca] channels) are expressed in podocytes. Here we show that [BK.sub.Ca] channels reciprocally coimmunoprecipitate with synaptopodin (Synpo) in mouse glomeruli, in mouse podocytes, and in a heterologous expression system (HEK293T cells) in which these proteins are transiently expressed. Synpo and Slo1 colocalize along the surface of the glomerular basement membrane in mouse glomeruli. Synpo interacts with [BK.sub.Ca] channels at COOH-terminal domains that overlap with an actin-binding domain on the channel molecule that is necessary for trafficking of [BK.sub.Ca] channels to the cell surface. Moreover, addition of exogenous [beta]-actin to mouse podocyte lysates reduces [BK.sub.Ca]-Synpo interactions. Coexpression of Synpo increases steady-state surface expression of [BK.sub.Ca] channels in HEK293T cells. However, Synpo does not affect the stability of cell surface [BK.sub.Ca] channels, suggesting a primary effect on the rate of forward trafficking, and Synpo coexpression does not affect [BK.sub.Ca] gating. Conversely, stable knockdown of Synpo expression in mouse podocyte cell lines reduces steady-state surface expression of [BK.sub.Ca] channels but does not affect total expression of [BK.sub.Ca] channels or their gating. The effects of Synpo on surface expression of [BK.sub.Ca] are blocked by inhibition of Rho signaling in HEK293T cells and in podocytes. Functional cell surface [BK.sub.Ca] channels in podocytes are also reduced by sustained (2 h) but not acute (15 min) depolymerization of actin with cytochalasin D. Synpo may regulate [BK.sub.Ca] channels through its effects on actin dynamics and by modulating interactions between [BK.sub.Ca] channels and regulatory proteins of the podocyte slit diaphragm. potassium channels; slit diaphragm; traffic; renal glomerulus; cytoskeleton doi: 10.1152/ajprenal.00206.2010.