The Role of K⁺ and Cl^-��Channels in the Regulation of Retinal Arteriolar Tone and Blood Flow

Purpose: This study tested the role of K(+)- and Cl(-)-channels in retinal arteriolar smooth muscle in the regulation of retinal blood flow. <br/><br/>Methods: Studies were carried out in adult Male Hooded Lister rats. Selectivity of ion channel blockers was established using electrophysiological recordings from smooth muscle in isolated arterioles under voltage clamp conditions. Leukocyte velocity and retinal arteriolar diameters were measured in anesthetised animals using leukocyte fluorography and fluorescein angiography imaging with a confocal scanning laser ophthalmoscope. These values were used to estimate volumetric flow, which was compared between control conditions and following intravitreal injections of ion channel blockers, either alone or in combination with the vasoconstrictor potent Endothelin 1 (Et1). <br/><br/>Results: Voltage activated K(+)-current (IKv) was inhibited by correolide, large conductance (BK) Ca(2+)-activated K(+)-current (IKCa) by Penitrem A, and Ca(2+)-activated Cl(-)-current (IClCa) by disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Intravitreal injections (10��l) of DIDS (estimated intraocular concentration 10mM) increased flow by 22%, whereas the BK-blockers Penitrem A (1��M) and iberiotoxin (4��M), and the IKv-inhibitor correolide (40��M) all decreased resting flow by approximately 10%. Et1 (104nM) reduced flow by almost 65%. This effect was completely reversed by DIDS but was unaffected by Penitrem A, iberiotoxin or correolide. <br/><br/>Conclusions: These results suggest that Cl(-)-channels in retinal arteriolar smooth muscle limit resting blood flow and play an obligatory role in Et1 responses. K(+)-channel activity promotes basal flow but exerts little modifying effect on the Et1 response. Cl(-)-channels may be appropriate molecular targets in retinal pathologies characterised by increased Et1 activity and reduced blood flow.

Implication des canaux K+ dans les processus de r��paration de l�����pith��lium respiratoire sain et fibrose kystique

La pathologie de la fibrose kystique (FK) est caus��e par des mutations du g��ne codant pour le canal Cl- CFTR. Au niveau respiratoire, cette dysfonction du transport trans��pith��lial de Cl- occasionne une alt��ration de la composition et du volume du liquide de surface des voies a��riennes. Une accumulation de mucus d��shydrat�� favorise alors la colonisation bact��rienne et une r��ponse inflammatoire chronique, entra��nant des l��sions ��pith��liales s��v��res au niveau des voies a��riennes et des alv��oles pouvant culminer en d��faillance respiratoire. Le principal objectif de mon projet de ma��trise ��tait d�����tudier les processus de r��paration de l�����pith��lium alv��olaire sain, l�����pith��lium bronchique sain et FK �� l���aide d���un mod��le in vitro de plaies m��caniques. Nos r��sultats d��montrent la pr��sence d���une boucle autocrine EGF/EGFR contr��lant les processus de migration cellulaire et de r��paration des l��sions m��caniques. D���autre part, nos exp��riences montrent que l���EGF stimule l���activit�� et l���expression des canaux K+ KATP, KvLQT1 et KCa3.1 des cellules ��pith��liales respiratoires. L���activation de ces canaux est cruciale pour les processus de r��paration puisque la majeure partie de la r��paration stimul��e �� l���EGF est abolie en pr��sence d���inhibiteurs de ces canaux. Nous avons ��galement observ�� que les cellules FK pr��sentent un d��lai de r��paration, probablement caus�� par un d��faut de la r��ponse EGF/EGFR et une activit��/expression r��duite des canaux K+. Nos r��sultats permettent de mieux comprendre les m��canismes de r��gulation des processus de r��paration de l�����pith��lium sain et FK. De plus, ils ouvrent de nouvelles options th��rapeutiques visant �� promouvoir, �� l���aide d���activateurs de canaux K+ et de facteurs de croissance, la r��g��n��ration de l�����pith��lium respiratoire chez les patients atteints de FK.

Implications des canaux K+ sur la r��gulation g��nique du canal ENaC, et impact de l'hyperglyc��mie sur le transport ionique et la r��paration de l'��pith��lium respiratoire

Dans mon projet de doctorat, j���ai ��tudi�� des fonctions primordiales de l�����pith��lium respiratoire telles que la r��gulation du transport ionique, la clairance liquidienne et la r��paration ��pith��liale. J���ai particuli��rement mis l���emphase sur le r��le des canaux potassiques qui interviennent dans ces trois fonctions de l�����pith��lium respiratoire. J���ai tout d���abord prouv�� que la modulation des canaux potassiques r��gulait l���activit�� du promoteur de ��ENaC, en partie via la voie de signalisation ERK1/2, dans des cellules alv��olaires. Cette r��gulation entra��ne une variation de l���expression g��nique et prot��ique du canal ENaC. Physiologiquement, il en r��sulte une augmentation du ph��nom��ne de clairance liquidienne suite �� l���activation des canaux K+, tandis que l���inhibition de ces canaux la diminue s��v��rement. J���ai aussi pu d��montrer que l���absence de canal KvLQT1 entra��nait une diminution du courant (ENaC) sensible �� l���amiloride, dans les cellules de trach��e en culture primaire, isol��es de souris KO pour kcnq1. Dans la seconde partie de mon ��tude, j���ai ��valu�� l���impact de l���hyperglyc��mie sur la capacit�� de transport ionique et de r��paration de cellules ��pith��liales bronchiques saines ou Fibrose Kystique. Mes r��sultats montrent que l���hyperglyc��mie diminue le transport trans��pith��lial de chlore et le transport basolat��ral de potassium. Des ��tudes pr��alables du laboratoire ayant montr�� que les canaux K+ et Cl- contr��lent les processus de r��paration, j���ai donc ��valu�� si ceux-ci ��taient modifi��s par l���hyperglyc��mie. Et en effet, l���hyperglyc��mie ralentit la vitesse de r��paration des cellules issues des voies a��riennes (CFBE-wt et CFBE-��F508). J���ai donc d��montr�� que le transport de potassium intervenait dans des fonctions cl��s de l�����pith��lium respiratoire, comme dans la r��gulation g��nique de canaux ioniques, le contr��le de la clairance liquidienne alv��olaire, et que l���hyperglyc��mie diminuait le transport ionique (K+ et Cl-) et la r��paration ��pith��liale.

��tudes de type structure fonction des mutations causant l���ataxie ��pisodique de type I sur les canaux potassiques d��pendants du voltage

Les ataxies ��pisodiques (EA) d���origine g��n��tique sont un groupe de maladies poss��dant un ph��notype et g��notype h��t��rog��nes, mais ont en commun la caract��ristique d���un dysfonctionnement c��r��belleux intermittent. Les EA de type 1 et 2 sont les plus largement reconnues des ataxies ��pisodiques autosomiques dominantes et sont caus��es par un dysfonctionnement des canaux ioniques voltage-d��pendants dans les neurones. La pr��sente ��tude se concentrera sur les mutations causant l'EA-1, retrouv��es dans le senseur de voltage (VSD) de Kv1.1, un canal tr��s proche de la famille des canaux Shaker. Nous avons caract��ris�� les propri��t��s ��lectrophysiologiques de six mutations diff��rentes �� la position F244 et partiellement celles des mutations T284 A/M, R297 K/Q/A/H, I320T, L375F, L399I et S412 C/I dans la s��quence du Shaker gr��ce �� la technique du ������cut open voltage clamp������ (COVC). Les mutations de la position F244 situ��es sur le S1 du canal Shaker sont caract��ris��es par un d��calement des courbes QV et GV vers des potentiels d��polarisants et modifient le couplage fonctionnel entre le domaine VSD et le pore. Un courant de fuite est observ�� durant la phase d'activation des courants transitoires et peut ��tre ��limin�� par l'application du 4-AP (4-aminopyridine) ou la r��insertion de l'inactivation de type N mais pas par le TEA (t��tra��thylamonium). Dans le but de mieux comprendre les m��canismes mol��culaires responsables de la stabilisation d���un ��tat interm��diaire, nous avons ��tudi�� s��par��ment la neutralisation des trois premi��res charges positives du S4 (R1Q, R2Q et R3Q). Il en est ressorti l���existence d���une interaction entre R2 et F244. Une seconde interface entre S1 et le pore proche de la surface extracellulaire agissant comme un second point d'ancrage et responsable des courants de fuite a ��t�� mis en lumi��re. Les r��sultats sugg��rent une anomalie du fonctionnement du VSD emp��chant la repolarisation normale de la membrane des cellules nerveuses affect��es �� la suite d'un potentiel d'action.

Anesthetic Activation of Central Respiratory Chemoreceptor Neurons Involves Inhibition of a THIK-1-Like Background K(+) Current

At surgical depths of anesthesia, inhalational anesthetics cause a loss of motor response to painful stimuli (i.e., immobilization) that is characterized by profound inhibition of spinal motor circuits. Yet, although clearly depressed, the respiratory motor system continues to provide adequate ventilation under these same conditions. Here, we show that isoflurane causes robust activation of CO(2)/pH-sensitive, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) of the rodent brainstem, in vitro and in vivo. In brainstem slices from Phox2b-eGFP mice, the firing of pH-sensitive RTN neurons was strongly increased by isoflurane, independent of prevailing pH conditions. At least two ionic mechanisms contributed to anesthetic activation of RTN neurons: activation of an Na(+)-dependent cationic current and inhibition of a background K(+) current. Single-cell reverse transcription-PCR analysis of dissociated green fluorescent protein-labeled RTN neurons revealed expression of THIK-1 (TWIK-related halothane-inhibited K(+) channel, K(2P)13.1), a channel that shares key properties with the native RTN current (i.e., suppression by inhalational anesthetics, weak rectification, inhibition by extracellular Na(+), and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO(2) levels. In these animals, isoflurane transiently enhanced activity of the respiratory system, an effect that was most prominent at low levels of respiratory drive and mediated primarily by an increase in respiratory frequency. These data indicate that inhalational anesthetics cause activation of RTN neurons, which serve an important integrative role in respiratory control; the increased drive provided by enhanced RTN neuronal activity may contribute, in part, to maintaining respiratory motor activity under immobilizing anesthetic conditions.

K-ATP(+) Channels-Independent Analgesic Action of Crotalus durissus cumanensis venom

The effect was investigated of the K+ channel blocker, glibenclamide, on the ability of Crotalus durissus cumanensis venom (CDCM) to promote peripheral antinociception. This was measured by formalin-induced nociception in male Swiss mice. CDCM (200 and 300 mu g/kg) produced an antinociceptive effect during phase 2 in the formalin test. The effect of CDCM (200 mu g/kg) was unaffected by the ATP-sensitive K+ channel blocker glibenclamide (2 mg/kg). These results suggest that CDCM is effective against acute pain. However, the ATP-sensitive K+ channels pathway is not contributable to the antinoeiceptive mechanism of CDCM.

Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter's syndrome and in adaptation to a high-K diet

Type II Bartter's syndrome is a hereditary hypokalemic renal salt-wasting disorder caused by mutations in the ROMK channel (Kir1.1; Kcnj1), mediating potassium recycling in the thick ascending limb of Henle's loop (TAL) and potassium secretion in the distal tubule and cortical collecting duct (CCT). Newborns with Type II Bartter are transiently hyperkalemic, consistent with loss of ROMK channel function in potassium secretion in distal convoluted tubule and CCT. Yet, these infants rapidly develop persistent hypokalemia owing to increased renal potassium excretion mediated by unknown mechanisms. Here, we used free-flow micropuncture and stationary microperfusion of the late distal tubule to explore the mechanism of renal potassium wasting in the Romk-deficient, Type II Bartter's mouse. We show that potassium absorption in the loop of Henle is reduced in Romk-deficient mice and can account for a significant fraction of renal potassium loss. In addition, we show that iberiotoxin (IBTX)-sensitive, flow-stimulated maxi-K channels account for sustained potassium secretion in the late distal tubule, despite loss of ROMK function. IBTX-sensitive potassium secretion is also increased in high-potassium-adapted wild-type mice. Thus, renal potassium wasting in Type II Bartter is due to both reduced reabsorption in the TAL and K secretion by max-K channels in the late distal tubule. �� 2006 International Society of Nephrology.

The peripheral L-arginine-nitric oxide-cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats

Crotalphine, a 14 amino acid peptide first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces a peripheral long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. In the present study, we further characterized the molecular mechanisms involved in this effect, determining the type of opioid receptor responsible for this effect and the involvement of the nitric oxide-cyclic GMP pathway and of K+ channels. Crotalphine (0.2 or 5 mu g/kg, orally; 0.0006 mu g/paw), administered on day 14 after nerve constriction, inhibited mechanical hyperalgesia and low-threshold mechanical allodynia. The effect of the peptide was antagonized by intraplantar administration of naltrindole, an antagonist of delta-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of kappa-opioid receptors. The effect of crotalphine was also blocked by 7-nitroindazole, an inhibitor of the neuronal nitric oxide synthase; by 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of guanylate cyclase activation; and by glibenclamide, an ATP-sensitive K+ channel blocker. The results suggest that peripheral delta-opioid and kappa-opioid receptors, the nitric oxide-cyclic GMP pathway, and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine. The present data point to the therapeutic potential of this peptide for the treatment of chronic neuropathic pain. Behavioural Pharmacology 23:14-24 (C) 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Ether-A -go-go 1 (Eag1) Potassium Channel Expression in Dopaminergic Neurons of Basal Ganglia is Modulated by 6-Hydroxydopamine Lesion

The ether A go-go (Eag) gene encodes the voltage-gated potassium (K+) ion channel Kv10.1, whose function still remains unknown. As dopamine may directly affect K+ channels, we evaluated whether a nigrostriatal dopaminergic lesion induced by the neurotoxin 6-hydroxydopamine (6-OHDA) would alter Eag1-K+ channel expression in the rat basal ganglia and related brain regions. Male Wistar rats received a microinjection of either saline or 6-OHDA (unilaterally) into the medial forebrain bundle. The extent of the dopaminergic lesion induced by 6-OHDA was evaluated by apomorphine-induced rotational behavior and by tyrosine hydroxylase (TH) immunoreactivity. The 6-OHDA microinjection caused a partial or complete lesion of dopaminergic cells, as well as a reduction of Eag1+ cells in a manner proportional to the extent of the lesion. In addition, we observed a decrease in TH immunoreactivity in the ipsilateral striatum. In conclusion, the expression of the Eag1-K+-channel throughout the nigrostriatal pathway in the rat brain, its co-localization with dopaminergic cells and its reduction mirroring the extent of the lesion highlight a physiological circuitry where the functional role of this channel can be investigated. The Eag1-K+ channel expression in dopaminergic cells suggests that these channels are part of the diversified group of ion channels that generate and maintain the electrophysiological activity pattern of dopaminergic midbrain neurons.

Nerve excitability studies characterize Kv1.1 fast potassium channel dysfunction in patients with episodic ataxia type 1

Episodic ataxia type 1 is a neuronal channelopathy caused by mutations in the KCNA1 gene encoding the fast K(+) channel subunit K(v)1.1. Episodic ataxia type 1 presents with brief episodes of cerebellar dysfunction and persistent neuromyotonia and is associated with an increased incidence of epilepsy. In myelinated peripheral nerve, K(v)1.1 is highly expressed in the juxtaparanodal axon, where potassium channels limit the depolarizing afterpotential and the effects of depolarizing currents. Axonal excitability studies were performed on patients with genetically confirmed episodic ataxia type 1 to characterize the effects of K(v)1.1 dysfunction on motor axons in vivo. The median nerve was stimulated at the wrist and compound muscle action potentials were recorded from abductor pollicis brevis. Threshold tracking techniques were used to record strength-duration time constant, threshold electrotonus, current/threshold relationship and the recovery cycle. Recordings from 20 patients from eight kindreds with different KCNA1 point mutations were compared with those from 30 normal controls. All 20 patients had a history of episodic ataxia and 19 had neuromyotonia. All patients had similar, distinctive abnormalities: superexcitability was on average 100% higher in the patients than in controls (P < 0.00001) and, in threshold electrotonus, the increase in excitability due to a depolarizing current (20% of threshold) was 31% higher (P < 0.00001). Using these two parameters, the patients with episodic ataxia type 1 and controls could be clearly separated into two non-overlapping groups. Differences between the different KCNA1 mutations were not statistically significant. Studies of nerve excitability can identify K(v)1.1 dysfunction in patients with episodic ataxia type 1. The simple 15 min test may be useful in diagnosis, since it can differentiate patients with episodic ataxia type 1 from normal controls with high sensitivity and specificity.

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories���hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.

Site-specific, photochemical proteolysis applied to ion channels in���vivo

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the ���signature��� Cys128���Cys142 disulfide loop of the nAChR �� subunit.

Ceramide-induced inhibition of T lymphocyte voltage-gated potassium channel is mediated by���tyrosine���kinases

The n-type K+ channel (n-K+, Kv1.3) in lymphocytes has been recently implicated in the regulation of Fas-induced programmed cell death. Here, we demonstrate that ceramide, a lipid metabolite synthesized upon Fas receptor ligation, inhibits n-K+ channel activity and induces a tyrosine phosphorylation of the Kv1.3 protein in Jurkat T lymphocytes. Tyrosine phosphorylation of the n-K+ channel correlated with an activation of the Src-like tyrosine kinase p56lck upon cellular treatment with the ceramide analog C6-ceramide. Because genetic deficiency of p56lck or inhibition of Src-like tyrosine kinases by herbimycin A prevented ceramide-mediated n-K+ channel inhibition and tyrosine phosphorylation, we propose a ceramide-initiated activation of p56lck resulting in tyrosine phosphorylation and inhibition of the n-K+ channel protein.

ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces���cerevisiae

A K+ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K+ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K+ transport capacity on yeast cells when heterologously expressed. In Xenopus laevis oocytes, expression yields an ungated K+-selective current whose attributes resemble the ���leak��� conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive. The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K+ channel family. Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals.

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in���MDCK���cells

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the ���small-conductance��� basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.