The 2-Pore Domain Potassium Channel TREK-1 Regulates Cytokine Secretion from Human Alveolar Epithelial Cells Independently of Potassium Currents

Tuesday, March 1, 2016 impaired gating modulation of Kv7.1 induced by KCNE1; leading to a shortening of the cardiac action potential. 2216-Pos Board B360 Molecular Dynamics Simulations of Mutant K Channels Involved in Severe Early Onset Epilepsy Robert A. Farley1, Yi Shi1, Yibo Wang2, Van Ngo2, Sergei Noskov2. 1 Physiology & Biophysics, University of Southern California, Los Angeles, CA, USA, 2Centre for Molecular Simulations, University of Calgary, Calgary, AB, Canada. Torkamani et al. (2014) identified three missense mutations in Kv1.2 channels as the genetic locus of severe early onset epilepsy in a cohort of affected patients. The mutant channels are characterized by the loss of K/Na ion selectivity and by the gain of a depolarizing cation conductance. Two of the three mutations in these Kv1.2 channels occur in amino acids that are located either in the selectivity filter of the channel or just outside the selectivity filter. The third mutation occurs in a region near the voltage sensor of the Kv1.2 channel. In order to understand the mechanistic basis for the altered channel phenotype caused by the mutations near the selectivity filter, we performed both equilibrium and non-equilibrium molecular dynamics (MD) simulations of Na and K movement through wild type and mutant KcsA channels. The amino acid sequence of the selectivity filter and nearby amino acids is the same in Kv1.2 and KcsA channels. In the non-equilibrium simulations, two ions of each type were pulled through the channels at constant velocity in both inward and outward directions. These simulations yielded both work distributions and free energy profiles for the movement of the ions through the channels. The equilibrium MD simulations were done using umbrella sampling to compute twodimensional PMF landscapes for both ionic species. The results of the simulations in the wild type and mutant channels offer clues about the mechanism of hyperexcitability associated with the epilepsy phenotype in neurons of patients with this disorder. 2217-Pos Board B361 A Missense Mutation in the Selectivity Filter of BK Affects the Channel’s Potassium Conductance Joa˜o L. Carvalho-de-Souza1, Tomoya Kubota1, Xiaofei Du2, Ramon Latorre3, Christopher M. Gomez2, Francisco Bezanilla1. 1 Biochem and Mol Biology, The University of Chicago, Chicago, IL, USA, 2 Department of Neurology, The University of Chicago, Chicago, IL, USA, 3 Centro Interdisciplinario de Neurociencia de Valparaı´so, Universidad de Valparaiso, Valparaiso, Chile. Large-conductance Ca2þ activated potassium channels (BK), which are synergistically modulated by voltage and intracellular calcium, play important role in the control of the cell excitability. Here we report data from a de novo missense mutation in KCNMA1, encoding the BK channel a subunit found in a patient with progressive cerebellar ataxia. The mutation changes the residue of glycine 354, in the selectivity filter signature sequence 352TVGYG-356 in potassium channels, into a serine. We expressed BK and G354S mutant BK (BK-G354S) channels in Xenopus oocytes and we analyzed macroscopic and on-cell single channel potassium currents recorded under voltage clamp. The macroscopic potassium currents are reduced in magnitude by about ten times and are slowly activated in oocytes expressing BK-G354S channels when compared with oocytes injected with cRNA for BK channels. The microscopic single channel conductance in BK-G354S is also depressed to about a tenth of the single channel conductance of BK channels, which suggest that the smaller macroscopic currents recorded with BK-G354S channels is not a mere problem of expression in the membrane. Actually, as revealed by expressing both proteins in mammalian cells and measuring their expression level using immunofluorescence techniques, BK-G354S channels are expressed in comparable levels with the expression of the wild-type BK channels. The mutation G354S in BK channel significantly decreases its potassium conductance, which importantly denotes a phenotype with loss of function. Biophysical data presented here provides crucial information that helps understanding the neurologic pathophysiology in the affected patient and it may also help in designing possible treatments for this and other similar cases. Supported by NIH GM030376, NS09466501 and NS082788-02. 2218-Pos Board B362 Pharmacology and Regulation of Fungal K2P Channels Ryan W. Manville1, Fernanda S. Povreslo2, Andrew Corran3, Anthony Lewis1. 1 Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom, 2Pharmacy, University of Campinas, Sao Paulo, Brazil, 3 Fungicide Biochemistry, Syngenta Limited, Bracknell, United Kingdom.

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Fungal K2P channels, or TOK (Two-pore Outwardly rectifying Kþ) channels, are a family of structurally and functionally unique Kþ channels with no known homologues in animals or plants. Activation of TOK channels leads to ion dyshomeostasis and fungal cell death, but little is known about their pharmacology. Here, using two-electrode voltage clamp of Xenopus laevis oocytes, we study the effects of human K2P channel (hK2P) modulators on two different fungal K2P channels cloned from human and wheat pathogens; CaTOK (Candida albicans) and MgTOK (Zymoseptoria Tritici). CaTOK and MgTOK currents were insensitive to the hK2P channel activator riluzole (100 mM) and hK2P inhibitor fluoxetine (100 mM), but exhibited augmented outwardly rectifying Kþ currents when exposed to arachidonic acid (AA) and the non-selective cyclooxygenase inhibitor BL-1249, both known hK2P channel activators. At 0 mV, 100 mM AA increased MgTOK currents by ~116% and CaTOK by ~155%, whereas 100 mM BL-1249 increased MgTOK currents by ~290% and CaTOK by ~100%. Both AA and BL-1249 induced significant leftward shifts in the voltage of half maximal activation (V½) of MgTOK (DV½ = ~40 mV and ~30 mV respectively) and CaTOK (DV½ = ~15 mV and ~9 mV respectively). Furthermore, AA and BL-1249 also increased the rate of current activation, but were without effect on deactivation kinetics. Both CaTOK and MgTOK were insensitive to the nonselective COX inhibitor aspirin (100 mM), suggesting that their regulation by BL-1249 does not occur through the accumulation of AA via COX inhibition. The findings are suggestive of shared regulatory mechanisms between fungal K2P channels and their human counterparts, and highlights the need for targeted therapeutics to counter fungal infections in animals and plants. 2219-Pos Board B363 The 2-Pore Domain Potassium Channel TREK-1 Regulates Cytokine Secretion from Human Alveolar Epithelial Cells Independently of Potassium Currents Andreas Schwingshackl1, Bin Teng2, Marc Borsotto3, Christopher M. Waters2. 1 Pediatrics, UCLA, Los Angeles, CA, USA, 2Physiology, University of Tennessee, Memphis, TN, USA, 3Institut de Pharmacologie Mole´culaire et Cellulair, Valbonne, France. We previously identified the 2-pore domain potassium (K2P) channel TREK-1 as an important regulator of tumor necrosis factor-a (TNF-a)-induced interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) secretion from alveolar epithelial cells (AECs). Furthermore, in an in vivo model of Acute Lung Injury (ALI) we found that TREK-1 deficiency led to increased lung damage but decreased broncho-alveolar lavage cytokine levels. Unfortunately, the mechanisms underlying TREK-1 regulation of inflammatory cytokine secretion remain poorly understood. This study was designed to determine whether the changes in cytokine secretion from TREK-1 deficient cells are due to the absence of TREK-1 protein or the lack of TREK-1-mediated potassium currents. For this purpose, we stimulated control and TREK-1 deficient human AECs (A549) with TNF-a and measured IL-6 and MCP-1 release by ELISA in the presence and absence of three TREK-1 blockers, spadin (0.5mM), amlodipine (2mM) and charybdotoxin (100nM). While spadin is recognized as a specific TREK-1 inhibitor, amlodipine and charybdotoxin at these concentrations also inhibit TREK-1. We found that TREK-1 deficient AECs released less IL-6 and more MCP-1 upon TNF-a stimulation but this cytokine release was unaffected by any of the three TREK-1 blockers. To determine whether cytokine secretion occurred independently of potassium currents in general, we raised the extracellular potassium concentration (100mM) to minimize the electrochemical driving force for potassium. Interestingly, we found that TNF-a-induced IL-6 and MCP-1 secretion were significantly impaired in the absence of a potassium gradient. In summary, TREK-1 protein, but not TREK-1 mediated potassium current, is required for IL-6 and MCP-1 secretion from AECs, although other potassium conductances appear important. These data suggest that TREK-1 may alter cytokine secretion from AECs by functioning as a regulatory molecule rather than a potassium channel. 2220-Pos Board B364 Calcium Current Properties and Leak Conductance in Mouse Muscle Fibers Overexpressing a Type 1 Hypokalemic Periodic Paralysis Mutant L-Type Calcium Channel Clarisse Fuster, Jimmy Perrot, Christine Berthier, Vincent Jacquemond, Bruno Allard. UMR 5534 CNRS/UCBL, University Lyon 1, Villeurbanne, France. Missense mutations in the gene encoding the alpha1 subunit of the L-type calcium channel CaV1.1 induce type 1 Hypokalemic Periodic Paralysis