Intermediate-conductance Channels Research Articles

IKCa channels control breast cancer metabolism including AMPK-driven autophagy

Ca2+-activated K+ channels of intermediate conductance (IK) are frequently overexpressed in breast cancer (BC) cells, while IK channel depletion reduces BC cell proliferation and tumorigenesis. This raises the question, of whether and mechanistically how IK activity interferes with the metabolic activity and energy consumption rates, which are fundamental for rapidly growing cells. Using BC cells obtained from MMTV-PyMT tumor-bearing mice, we show that both, glycolysis and mitochondrial ATP-production are reduced in cells derived from IK-deficient breast tumors. Loss of IK altered the sub-/cellular K+- and Ca2+- homeostasis and mitochondrial membrane potential, ultimately resulting in reduced ATP-production and metabolic activity. Consequently, we find that BC cells lacking IK upregulate AMP-activated protein kinase activity to induce autophagy compensating the glycolytic and mitochondrial energy shortage. Our results emphasize that IK by modulating cellular Ca2+- and K+-dynamics contributes to the remodeling of metabolic pathways in cancer. Thus, targeting IK channel might disturb the metabolic activity of BC cells and reduce malignancy.

Possible new strategies for the treatment of congenital hyperinsulinism

Objective Congenital hyperinsulinism (CHI) is a rare disease characterized by persistent hypoglycemia as a result of inappropriate insulin secretion, which can lead to irreversible neurological defects in infants. Poor efficacy and strong adverse effects of the current medications impede successful treatment. The aim of the study was to investigate new approaches to silence β-cells and thus attenuate insulin secretion. Research design and methods In the scope of our research, we tested substances more selective and more potent than the gold standard diazoxide that also interact with neuroendocrine ATP-sensitive K+ (KATP) channels. Additionally, KATP channel-independent targets as Ca2+-activated K+ channels of intermediate conductance (KCa3.1) and L-type Ca2+ channels were investigated. Experiments were performed using human islet cell clusters isolated from tissue of CHI patients (histologically classified as pathological) and islet cell clusters obtained from C57BL/6N (WT) or SUR1 knockout (SUR1-/-) mice. The cytosolic Ca2+ concentration ([Ca2+]c) was used as a parameter for the pathway regulated by electrical activity and was determined by fura-2 fluorescence. The mitochondrial membrane potential (ΔΨ) was determined by rhodamine 123 fluorescence and single channel currents were measured by the patch-clamp technique. Results The selective KATP channel opener NN414 (5 µM) diminished [Ca2+]c in isolated human CHI islet cell clusters and WT mouse islet cell clusters stimulated with 10 mM glucose. In islet cell clusters lacking functional KATP channels (SUR1-/-) the drug was without effect. VU0071063 (30 µM), another KATP channel opener considered to be selective, lowered [Ca2+]c in human CHI islet cell clusters. The compound was also effective in islet cell clusters from SUR1-/- mice, showing that [Ca2+]c is influenced by additional effects besides KATP channels. Contrasting to NN414, the drug depolarized ΔΨ in murine islet cell clusters pointing to severe interference with mitochondrial metabolism. An opener of KCa3.1 channels, DCEBIO (100 µM), significantly decreased [Ca2+]c in SUR1-/- and human CHI islet cell clusters. To target L-type Ca2+ channels we tested two already approved drugs, dextromethorphan (DXM) and simvastatin. DXM (100 µM) efficiently diminished [Ca2+]c in stimulated human CHI islet cell clusters as well as in stimulated SUR1-/- islet cell clusters. Similar effects on [Ca2+]c were observed in experiments with simvastatin (7.2 µM). Conclusions NN414 seems to provide a good alternative to the currently used KATP channel opener diazoxide. Targeting KCa3.1 channels by channel openers or L-type Ca2+ channels by DXM or simvastatin might be valuable approaches for treatment of CHI caused by mutations of KATP channels not sensitive to KATP channel openers.

Treatment with senicapoc in a porcine model of acute respiratory distress syndrome

BackgroundSenicapoc is a potent and selective blocker of KCa3.1, a calcium-activated potassium channel of intermediate conductance. In the present study, we investigated whether there is a beneficial effect of senicapoc in a large animal model of acute respiratory distress syndrome (ARDS). The primary end point was the PaO2/FiO2 ratio.MethodsARDS was induced in female pigs (42–49 kg) by repeated lung lavages followed by injurious mechanical ventilation. Animals were then randomly assigned to vehicle (n = 9) or intravenous senicapoc (10 mg, n = 9) and received lung-protective ventilation for 6 h.ResultsFinal senicapoc plasma concentrations were 67 ± 18 nM (n = 9). Senicapoc failed to change the primary endpoint PaO2/FiO2 ratio (senicapoc, 133 ± 23 mmHg; vehicle, 149 ± 68 mmHg). Lung compliance remained similar in the two groups. Senicapoc reduced the level of white blood cells and neutrophils, while the proinflammatory cytokines TNFα, IL-1β, and IL-6 in the bronchoalveolar lavage fluid were unaltered 6 h after induction of the lung injury. Senicapoc-treatment reduced the level of neutrophils in the alveolar space but with no difference between groups in the cumulative lung injury score. Histological analysis of pulmonary hemorrhage indicated a positive effect of senicapoc on alveolar–capillary barrier function, but this was not supported by measurements of albumin content and total protein in the bronchoalveolar lavage fluid.ConclusionsIn summary, senicapoc failed to improve the primary endpoint PaO2/FiO2 ratio, but reduced pulmonary hemorrhage and the influx of neutrophils into the lung. These findings open the perspective that blocking KCa3.1 channels is a potential treatment to reduce alveolar neutrophil accumulation and improve long-term outcome in ARDS.

A KCa3.1 Channel Opener, ASP0819, Modulates Nociceptive Signal Processing from Peripheral Nerves in Fibromyalgia-Like Pain in Rats.

PurposeAlthough abnormal peripheral and central pain processing has been observed in fibromyalgia (FM) patients, the biomechanics and pathophysiology, surrounding the peripheral mechanism are not well understood. An intermediate conductance channel, KCa3.1, is expressed in peripheral sensory nerve fibers where it maintains the resting membrane potential and controls nerve firing, making it a plausible target for peripheral therapeutic interventions. ASP0819, a KCa3.1 channel opener, is an orally available molecular entity and is used in this investigation to elucidate the role of KCa3.1 in signal processing of pain in FM.MethodsHuman or rat KCa3.1 channel-expressing cells were used for evaluating the main action of the compound. Effects of the compound on withdrawal behavior by mechanical stimulation were examined in reserpine-induced myalgia (RIM) and vagotomy-induced myalgia (VIM) models of rats. In addition, in vivo electrophysiological analysis was performed to examine the peripheral mechanisms of action of the compound. Other pain models were also examined.ResultsASP0819 increased the negative membrane potential in a concentration-dependent manner. Oral administration of ASP0819 significantly recovered the decrease in muscle pressure threshold in rat FM models of RIM and VIM. The in vivo electrophysiological experiments showed that Aδ- and C-fibers innervating the leg muscles in the RIM model demonstrated increased spontaneous and mechanically evoked firing compared with normal rats. Intravenous infusion of ASP0819 significantly reduced both the spontaneous activity and mechanically evoked responses in Aδ-fibers in the rat RIM model. ASP0819 significantly reduced the number of abdominal contractions as an indicator of abdominal pain behaviors in the rat visceral extension model and withdrawal responses in the osteoarthritis model, respectively.ConclusionThese findings suggest that ASP0819 may be a promising analgesic agent with the ability to modulate peripheral pain signal transmission. Its use in the treatment of several pain conditions should be explored, chief amongst these being FM pain.

Possible New Strategies for the Treatment of Congenital Hyperinsulinism.

ObjectiveCongenital hyperinsulinism (CHI) is a rare disease characterized by persistent hypoglycemia as a result of inappropriate insulin secretion, which can lead to irreversible neurological defects in infants. Poor efficacy and strong adverse effects of the current medications impede successful treatment. The aim of the study was to investigate new approaches to silence β-cells and thus attenuate insulin secretion.Research Design and MethodsIn the scope of our research, we tested substances more selective and more potent than the gold standard diazoxide that also interact with neuroendocrine ATP-sensitive K+ (KATP) channels. Additionally, KATP channel-independent targets as Ca2+-activated K+ channels of intermediate conductance (KCa3.1) and L-type Ca2+ channels were investigated. Experiments were performed using human islet cell clusters isolated from tissue of CHI patients (histologically classified as pathological) and islet cell clusters obtained from C57BL/6N (WT) or SUR1 knockout (SUR1-/-) mice. The cytosolic Ca2+ concentration ([Ca2+]c) was used as a parameter for the pathway regulated by electrical activity and was determined by fura-2 fluorescence. The mitochondrial membrane potential (ΔΨ) was determined by rhodamine 123 fluorescence and single channel currents were measured by the patch-clamp technique.ResultsThe selective KATP channel opener NN414 (5 µM) diminished [Ca2+]c in isolated human CHI islet cell clusters and WT mouse islet cell clusters stimulated with 10 mM glucose. In islet cell clusters lacking functional KATP channels (SUR1-/-) the drug was without effect. VU0071063 (30 µM), another KATP channel opener considered to be selective, lowered [Ca2+]c in human CHI islet cell clusters. The compound was also effective in islet cell clusters from SUR1-/- mice, showing that [Ca2+]c is influenced by additional effects besides KATP channels. Contrasting to NN414, the drug depolarized ΔΨ in murine islet cell clusters pointing to severe interference with mitochondrial metabolism. An opener of KCa3.1 channels, DCEBIO (100 µM), significantly decreased [Ca2+]c in SUR1-/- and human CHI islet cell clusters. To target L-type Ca2+ channels we tested two already approved drugs, dextromethorphan (DXM) and simvastatin. DXM (100 µM) efficiently diminished [Ca2+]c in stimulated human CHI islet cell clusters as well as in stimulated SUR1-/- islet cell clusters. Similar effects on [Ca2+]c were observed in experiments with simvastatin (7.2 µM).ConclusionsNN414 seems to provide a good alternative to the currently used KATP channel opener diazoxide. Targeting KCa3.1 channels by channel openers or L-type Ca2+ channels by DXM or simvastatin might be valuable approaches for treatment of CHI caused by mutations of KATP channels not sensitive to KATP channel openers.

Structure, Gating and Basic Functions of the Ca2+-activated K Channel of Intermediate Conductance.

BackgroundThe KCa3.1 channel is the intermediate-conductance member of the Ca2+-activated K channel superfamily. It is widely expressed in excitable and non-excitable cells, where it plays a major role in a number of cell functions. This paper aims at illustrating the main structural, biophysical and modulatory properties of the KCa3.1 channel, and providing an account of experimental data on its role in volume regulation and Ca2+ signals.MethodsResearch and online content related to the structure, structure/function relationship, and physiological role of the KCa3.1 channel are reviewed.ResultsExpressed in excitable and non-excitable cells, the KCa3.1 channel is voltage independent, its opening being exclusively gated by the binding of intracellular Ca2+ to calmodulin, a Ca2+-binding protein constitutively associated with the C-terminus of each KCa3.1 channel α subunit. The KCa3.1 channel activates upon high affinity Ca2+ binding, and in highly coordinated fashion giving steep Hill functions and relatively low EC50 values (100-350 nM). This high Ca2+ sensitivity is physiologically modulated by closely associated kinases and phosphatases. The KCa3.1 channel is normally activated by global Ca2+ signals as resulting from Ca2+ released from intracellular stores, or by the refilling influx through store operated Ca2+ channels, but cases of strict functional coupling with Ca2+-selective channels are also found. KCa3.1 channels are highly expressed in many types of cells, where they play major roles in cell migration and death. The control of these complex cellular processes is achieved by KCa3.1 channel regulation of the driving force for Ca2+ entry from the extracellular medium, and by mediating the K+ efflux required for cell volume control.ConclusionMuch work remains to be done to fully understand the structure/function relationship of the KCa3.1 channels. Hopefully, this effort will provide the basis for a beneficial modulation of channel activity under pathological conditions.

SK4 channels modulate Ca2+ signalling and cell cycle progression in murine breast cancer.

Oncogenic signalling via Ca2+‐activated K+ channels of intermediate conductance (SK4, also known as KCa3.1 or IK) has been implicated in different cancer entities including breast cancer. Yet, the role of endogenous SK4 channels for tumorigenesis is unclear. Herein, we generated SK4‐negative tumours by crossing SK4‐deficient (SK4 KO) mice to the polyoma middle T‐antigen (PyMT) and epidermal growth factor receptor 2 (cNeu) breast cancer models in which oncogene expression is driven by the retroviral promoter MMTV. Survival parameters and tumour progression were studied in cancer‐prone SK4 KO in comparison with wild‐type (WT) mice and in a syngeneic orthotopic mouse model following transplantation of SK4‐negative or WT tumour cells. SK4 activity was modulated by genetic or pharmacological means using the SK4 inhibitor TRAM‐34 in order to establish the role of breast tumour SK4 for cell growth, electrophysiological signalling, and [Ca2+]i oscillations. Ablation of SK4 and TRAM‐34 treatment reduced the SK4‐generated current fraction, growth factor‐dependent Ca2+ entry, cell cycle progression and the proliferation rate of MMTV‐PyMT tumour cells. In vivo, PyMT oncogene‐driven tumorigenesis was only marginally affected by the global lack of SK4, whereas tumour progression was significantly delayed after orthotopic implantation of MMTV‐PyMT SK4 KO breast tumour cells. However, overall survival and progression‐free survival time in the MMTV‐cNeu mouse model were significantly extended in the absence of SK4. Collectively, our data from murine breast cancer models indicate that SK4 activity is crucial for cell cycle control. Thus, the modulation of this channel should be further investigated towards a potential improvement of existing antitumour strategies in human breast cancer.

Expression of a Diverse Array of Ca2+-Activated K+ Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney.

The voltage- and Ca2+-activated, large conductance K+ channel (BK, maxi-K) is expressed in the collecting duct system of kidney where it underlies flow- and Ca2+-dependent K+ excretion. To determine if other Ca2+-activated K+ channels (KCa) may participate in this process, mouse kidney and the K+-secreting mouse cortical collecting duct (CCD) cell line, mCCDcl1, were assessed for TRPV4 and KCa channel expression and cross-talk. qPCR mRNA analysis and immunocytochemical staining demonstrated TRPV4 and KCa expression in mCCDcl1 cells and kidney connecting tubule (CNT) and CCD. Three subfamilies of KCa channels were revealed: the high Ca2+-binding affinity small-conductance SK channels, SK1and SK3, the intermediate conductance channel, IK1, and the low Ca2+-binding affinity, BK channel (BKα subunit). Apparent expression levels varied in CNT/CCD where analysis of CCD principal cells (PC) and intercalated cells (IC) demonstrated differential staining: SK1:PC<IC, and SK3:PC>IC, IK1:PC>IC, BKα:PC = IC, and TRPV4:PC>IC. Patch clamp analysis and fluorescence Ca2+ imaging of mCCDcl1 cells demonstrated potent TRPV4-mediated Ca2+ entry and strong functional cross-talk between TRPV4 and KCa channels. TRPV4-mediated Ca2+ influx activated each KCa channel, as evidenced by selective inhibition of KCa channels, with each active KCa channel enhancing Ca2+ entry (due to membrane hyperpolarization). Transepithelial electrical resistance (TEER) analysis of confluent mCCDcl1 cells grown on permeable supports further demonstrated this cross-talk where TRPV4 activation induce a decrease in TEER which was partially restored upon selective inhibition of each KCa channel. It is concluded that SK1/SK3 and IK1 are highly expressed along with BKα in CNT and CCD and are closely coupled to TRPV4 activation as observed in mCCDcl1 cells. The data support a model in CNT/CCD segments where strong cross talk between TRPV4-mediated Ca2+ influx and each KCa channel leads to enhance Ca2+ entry which will support activation of the low Ca2+-binding affinity BK channel to promote BK-mediated K+ secretion.

Contribution of the KCa3.1 channel-calmodulin interactions to the regulation of the KCa3.1 gating process.

The Ca2+-activated potassium channel of intermediate conductance, KCa3.1, is now emerging as a therapeutic target for a large variety of health disorders. The Ca2+ sensitivity of KCa3.1 is conferred by the Ca2+-binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C terminus. It was proposed on the basis of the crystal structure obtained for the C-terminal region of the rat KCa2.2 channel (rSK2) with CaM that the binding of Ca2+ to the CaM N-lobe results in CaM interlocking the C-terminal regions of two adjacent KCa3.1 subunits, leading to the formation of a dimeric structure. A study was thus undertaken to identify residues of the CaM N-lobe–KCa3.1 complex that either contribute to the channel activation process or control the channel open probability at saturating Ca2+ (Pomax). A structural homology model of the KCa3.1–CaM complex was first generated using as template the crystal structure of the C-terminal region of the rat KCa2.2 channel with CaM. This model was confirmed by cross-bridging residues R362 of KCa3.1 and K75 of CaM. Patch-clamp experiments were next performed, demonstrating that the solvation energy of the residue at position 367 in KCa3.1 is a key determinant to the channel Pomax and deactivation time toff. Mutations of residues M368 and Q364 predicted to form anchoring points for CaM binding to KCa3.1 had little impact on either toff or Pomax. Finally, our results show that channel activation depends on electrostatic interactions involving the charged residues R362 and E363, added to a nonpolar energy contribution coming from M368. We conclude that electrostatic interactions involving residues R362 and E363 and hydrophobic effects at M368 play a prominent role in KCa3.1 activation, whereas hydrophobic interactions at S367 are determinant to the stability of the CaM–KCa3.1 complex throughout gating.

KCa3.1 channels maintain endothelium‐dependent vasodilatation in isolated perfused kidneys of spontaneously hypertensive rats after chronic inhibition of NOS

The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury. Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar-Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with N(ω) -nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg(-1) ·day(-1) ). Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of N(w) -nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca(2+) -activated K(+) channels of intermediate conductance (K(Ca) 3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca(2+) -activated K(+) channels of small conductance (K(Ca) 2.3), was ineffective. Dilatation induced by an opener of K(Ca) 3.1/K(Ca) 2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for K(Ca) 3.1, but not K(Ca) 2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR). In SHR kidneys, an EDHF-mediated response, involving activation of K(Ca) 3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability.

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells

Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca(2+)-activated K(+) channels are known to control spike frequency in central neurons, Ca(2+)-activated K(+) channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca(2+) channels at the nanodomain level to support a previously undescribed transient voltage- and Ca(2+)-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca(2+) influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.

Swelling-Activated Anion Channels Are Essential for Volume Regulation of Mouse Thymocytes

Channel-mediated trans-membrane chloride movement is a key process in the active cell volume regulation under osmotic stress in most cells. However, thymocytes were hypothesized to regulate their volume by activating a coupled K-Cl cotransport mechanism. Under the patch-clamp, we found that osmotic swelling activates two types of macroscopic anion conductance with different voltage-dependence and pharmacology. At the single-channel level, we identified two types of events: one corresponded to the maxi-anion channel, and the other one had characteristics of the volume-sensitive outwardly rectifying (VSOR) chloride channel of intermediate conductance. A VSOR inhibitor, phloretin, significantly suppressed both macroscopic VSOR-type conductance and single-channel activity of intermediate amplitude. The maxi-anion channel activity was largely suppressed by Gd3+ ions but not by phloretin. Surprisingly, [(dihydroindenyl)oxy] alkanoic acid (DIOA), a known antagonist of K-Cl cotransporter, was found to significantly suppress the activity of the VSOR-type single-channel events with no effect on the maxi-anion channels at 10 μM. The regulatory volume decrease (RVD) phase of cellular response to hypotonicity was mildly suppressed by Gd3+ ions and was completely abolished by phloretin suggesting a major impact of the VSOR chloride channel and modulatory role of the maxi-anion channel. The inhibitory effect of DIOA was also strong, and, most likely, it occurred via blocking the VSOR Cl− channels.

Triarylmethane‐34 (TRAM‐34), inhibitor of Ca2+‐activated K+ channels of intermediate conductance, is a potent blocker of gap junctions

Ca2+‐activated K+ channels of intermediate conductance (IKCa) and gap junctions (GJs) between endothelial and smooth muscle cells have been proposed to play a central role in the vasodilator response mediated by the endothelium‐derived hyperpolarizing factor (EDHF). Although TRAM‐34, the IKCa channels blocker, has emerged as an important pharmacological tool for studying EDHF‐mediated responses, its effect on GJs has not been described. We analyzed intercellular communication and cellular distribution of the GJ protein subunit, connexins (Cxs), in HeLa cells transfected with Cx43 or Cx43 conjugated to EGFP. Treatment with 10μM TRAM‐34 induced a time dependent reduction in dye coupling to Lucifer yellow; the average number of coupled cells observed in positive tests was reduced by ~50% after 20 min and abolished after 70 min. Dye coupling was recovered to control 30 min after TRAM‐34 wash out. The reduction in GJ communication was not associated with changes in levels or distribution of Cx43‐EGFP. Therefore, TRAM‐34, in addition to inhibit IKCa channels, also potently blocks the cell‐to‐cell communication mediated by the GJ protein Cx43 that is an important connexin involved in the coordination of resistance vessel function.VRAID 15/2009, FONDEF D07I1086.

Physiological significance of SK4 channels in pancreatic β-cell oscillations

Pancreatic beta-cells show oscillations in membrane potential (Vm), the cytosolic Ca2+ concentration ([Ca2+]c) and finally insulin secretion. It is well accepted that the initiation of a burst phase with action potentials is mediated by voltage-dependent Ca2+ (and Na+) channels. The mechanism triggering the onset of interburst phases is less clear. The exact nature of the K+ channels that hyperpolarize Vm to maintain the rhythmic activity is unknown. In 1999 Göpel and co-workers1 described a current termed Kslow and claimed that this current terminates the burst phases. KATP current is a part of the Kslow current. We could show that the Ca2+-dependent K+ current through K+ channels of intermediate conductance (SK4, KCa3.1 or IK1) also contributes to the Kslow current. We suggest that the Kslow current is composed of two currents through metabolism-regulated K+ channels, KATP (regulated by ATP) and SK4 (regulated by Ca2+). We further propose that the SK4 component of the Kslow current can trigger oscillations in mice without functioning KATP channels.

C014 Endothelial dysfunction in isolated perfused kidneys of L-NAME-treated spontaneous hypertensive rats

In hypertensive humans, the development of end-stage renal disease is often associated with an endothelial dysfunction attributed to a decrease in the bioavailability of nitric oxide (NO). An early endothelial dysfunction is also reported in most animal models of hypertension and profound alterations in renal function are observed in aged hypertensive animals. As kidneys play an important role in blood pressure regulation, the purpose of the present study was to determine whether a chronic treatment with a NO synthase inhibitor, L-NAME (Nω- nitro-L-arginine methyl ester) accelerates the renal dysfunction in younger adult spontaneously hypertensive rats (SHR). Indeed, in this model, the alterations in cardiac function mimic those observed in aging SHR. Male SHR were subjected to chronic L-NAME-treatment (6 mg/ kg/day) for two weeks. At 19 weeks, the kidneys were isolated, perfused with oxygenated physiological salt solution and changes in renal perfusion pressure continuously recorded. During a sustained constriction caused by methoxamine, bolus injections of acetylcholine and of the NO donor sodium nitroprusside caused dose-dependent dilatations in both groups of SHR. The responses to acetylcholine were not affected by the L-NAME treatment while those to sodium nitroprusside were significantly augmented. In the two groups, indomethacin, a cyclooxygenase inhibitor, did not affect the vasodilatation caused by acetylcholine. However, an acute treatment with another NO synthase inhibitor, L-nitroarginine, produced a major inhibition of the acetylcholine response in the SHR while it did not affect it in the kidney of SHR chronically treated with L-NAME. In the presence of indomethacin plus L-nitro-arginine, TRAM-34, an inhibitor of Ca2+-activated K+ channels of intermediate conductance, IK(Ca), blocked the responses to acetylcholine in both groups of SHR. These data show that in SHR, the acetylcholine-induced vasodilatation involves both NO release and an endothelium-derived hyperpolarizing factor (EDHF)-mediated component while in the L-NAME-treated SHR, the NO-component is abolished and the EDHF-mediated component becomes the major contributor to this vasodilatation. In the kidney of L-NAME-treated SHR, EDHF-mediated responses are a compensatory mechanism that sustain endothelium-dependent dilatations when NO activity is impaired. The responses involve IK(Ca), activation and must be further characterized.

Senicapoc (ICA-17043): a potential therapy for the prevention and treatment of hemolysis-associated complications in sickle cell anemia

Sickle cell disease (SCD) is characterized by hemolytic as well as vaso-occlusive complications. The development of treatments for this inherited disease is based on an understanding of its pathophysiology. Polymerization of sickle hemoglobin is dependent on several independent factors, including the intracellular hemoglobin concentration. The hydration state (and intracellular hemoglobin concentration) of the sickle erythrocyte depends on the loss of solute and osmotically obliged water through specific pathways. Senicapoc (also known as ICA-17043) is a potent blocker of the Gardos channel, a calcium-activated potassium channel of intermediate conductance, in the red blood cell. Preclinical studies and studies in transgenic models of SCD show that inhibition of potassium efflux through the Gardos channel is associated with an increased hemoglobin level, decreased dense cells and decreased hemolysis. Senicapoc is well tolerated when administered to SCD patients and produces dose-dependent increases in hemoglobin and decreases in markers of hemolysis. Despite the lack of a reduction in the frequency of pain episodes, the increasing recognition that hemolysis contributes to the development of several SCD-related complications suggests that by decreasing hemolysis, senicapoc may yet prove to be beneficial in this disease.

The effects of growth factors on hIK1 protein expression levels in transfected HEK293 cells

The calcium (Ca2+)‐activated potassium (K+) channel, hIK1, is a human intermediate conductance channel expressed in numerous epithelia linings including the placenta, lungs and gastrointestinal tract. The functional roles for hIK1 underline processes such as ion transport, volume regulation, cell growth and proliferation. To investigate hIK1's role in cell growth, we examined the effects of the growth factors: epidermal growth factor (EGF), fibroblast growth factor (FGF), and tumor necrosis growth factor alpha (TNF‐α) on hIK1 protein expression. The protein levels were measured by treating human embryonic kidney (HEK) cells transfected with hIK1. The growth factors were administered 24 hours prior to analysis. Cells were lysed, total protein was collected and equal quantities were run on SDS‐page gel. Antibodies were used to detect hIK1 expression. From the total isolated protein the relative thickness of the treated cell bands on the western blot were compared to the untreated hIK1‐HEK cell bands to determine if total hIK1 protein expression was increased or decreased. It was found that EGF‐treatment increased total hIK1 protein expression and TNF‐α treatment decreased hIK1 protein levels. These results suggest that growth factor interactions with hIK1 are mediating the channel's role in cell growth and proliferation.

Histidine Phosphorylation of the Potassium Channel KCa3.1 by Nucleoside Diphosphate Kinase B Is Required for Activation of KCa3.1 and CD4 T Cells

The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation.

Pharmacology of N ‐, L ‐, and B ‐subtypes of nematode nAChR resolved at the single‐channel level in Ascaris suum

Pharmacological experiments on Ascaris suum have demonstrated the presence of three (N-, L-, and B-) subtypes of cholinergic receptor mediating contraction of body wall muscle in parasitic nematodes. In the present study, these ionotropic acetylcholine (ACh) receptors (nAChRs) were activated by levamisole and bephenium under patch-clamp conditions and competitively antagonized by paraherquamide and 2-desoxoparaherquamide. A number of recordings exhibited three separate current amplitude levels, indicating the presence of small, intermediate, and large conductance subtypes of receptor. The mean conductance of the small conductance subtype, G25, was 22 +/- 1 pS; the intermediate conductance channel, G35, was 33 +/- 1 pS; and the large conductance channel, G45, was 45 +/- 1 pS. The small channel was not antagonized significantly by paraherquamide and was identified as the N-subtype. The intermediate channel was preferentially activated by levamisole rather than bephenium and antagonized by paraherquamide: the intermediate channel was identified as the L-subtype. The large conductance channel was preferentially activated by bephenium, antagonized more by 2-desoxoparaherquamde than by paraherquamide and was identified as the B-subtype. These observations reveal that the three channel subtypes have different selectivity for cholinergic anthelmintics. The different selectivity of these compounds should be considered when dealing with drug resistant infections.

P2Y2 and P2Y4 receptors regulate pancreatic Ca2+-activated K+ channels differently

Extracellular ATP is an important regulator of transepithelial transport in a number of tissues. In pancreatic ducts, we have shown that ATP modulates epithelial K+ channels via purinergic receptors, most likely the P2Y2 and P2Y4 receptors, but the identity of the involved K+ channels was not clear. In this study, we show by RT-PCR analysis that rat pancreatic ducts express Ca(2+)-activated K+ channels of intermediate conductance (IK) and big conductance (BK), but not small conductance (SK). Possible interactions between P2Y receptors and these Ca(2+)-activated K+ channels were examined in co-expression experiments in Xenopus laevis oocytes. K+ channel activity was measured electrophysiologically in oocytes stimulated with UTP (0.1 mM). UTP stimulation of oocytes expressing P2Y4 receptors and BK channels resulted in a 30% increase in the current through the expressed channels. In contrast, stimulation of P2Y2 receptors led to a 20% inhibition of co-expressed BK channel activity, a response that was sensitive to TEA. Furthermore, co-expression of IK channels with P2Y4 and P2Y2 receptors resulted in a large hyperpolarization and 22-fold and 5-fold activation of currents by UTP, respectively. Taken together, this study shows that there are different interactions between the subtypes of P2Y purinergic receptors and different Ca(2+)-activated K+ channels.