Regulation of a Fungal K2P Channel by Protein Kinases

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Monday, February 13, 2017

1247-Pos Board B315 S. aureus Sphingomyelinase is a State-Dependent Inhibitor of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Brandon Stauffer. Emory University, Atlanta, GA, USA. Therapeutics for the treatment of cystic fibrosis have recently progressed to include drugs that directly increase CFTR channel activity; however, the influence of bacterial virulence factors on the efficacy of these potentiators is not well understood. One secreted protein of particular interest is sphingomyelinase (SMase), a sphingomyelin-specific phosphodiesterase that generates the signaling lipid ceramide and has been shown previously to inhibit CFTR channel activity in Xenopus laevis oocytes and the immortalized Calu3 epithelial cell line. In this study we performed mechanistic experiments to better understand how bacterial sphingomyelinase impacts CFTR channel function and how that inhibition affects the efficacy of the clinically prescribed CFTR potentiator, Kalydeco (VX770). Our data shown that purified S. aureus SMase is a gating modifier of CFTR wherein channels are inhibited at the plasma membrane independent of the R-domain, the best understood regulatory domain of CFTR. We also found that inhibition occurs in channels that are isolated from the enzyme and does not occur in excised patches when SMase is backfilled into the pipette suggesting that inhibition occurs through a cytosolic signaling event. Consistent with the idea that SMase activity modulates CFTR gating at the membrane, we found that inhibition efficacy was dependent upon CFTR conformational state. Specifically, we found that mutations known to increase channel activity significantly decreased the rate of inhibition by SMase. Finally, we found evidence that inhibited channels cannot be rescued by applying VX770 suggesting that inhibited channels are effectively removed from the druggable pool. Taken together, our data suggest that S. aureus SMase is a gating modifier of CFTR that inhibits channel activity in a manner that renders VX770 ineffective. 1248-Pos Board B316 Mechanism of Action of a Small Molecule Activator of PhosphoinositideDependent GIRK Channels Yu Xu1, Takeharu Kawano1, Junghoon Ha2, Sumanta Garai1, Guoqing Xiang2, Ganesh Thakur1, Diomedes E. Logothetis1. 1 the Department of Pharmaceutical Sciences at the Bouve College of Health Sciences, Northeastern University, Boston, MA, USA, 2Department of Physiology & Biophysics, Virginia Commonwealth University, Richmond, VA, USA. Introduction: G protein-sensitive inwardly rectifying potassium channels (GIRK) are important integrators of G protein-coupled receptor (GPCR) signaling. Activation of the cardiac GIRK1 and GIRK4 heteromer (GIRK1/4) hyperpolarizes atrial myocytes, shortens the action potential, and slows heart rate. Loss of function mutations of GIRK1/4 channels have been found to cause congenital long QT syndrome LQT13. The first potent, GIRK1-containing, and neuron-selective small GIRK activator, ML297, could serve as an antiepileptic and anxiolytic drug promising potential therapeutic utility. Further studies identified that two unique amino acids Phe137 (F, in the pore helix) and Asp173 (D, in TM2) of GIRK1 are necessary and sufficient for the activation of GIRK channels by ML297. This result also suggests, unlike other cytosolic activators (Gbg, Naþ, and ethanol), ML297 may act in the channel’s transmembrane domains, activating the channel by a distinct mechanism. Here we investigate the ML297 binding site(s) in FD containing GIRK (GIRKFD) channel and its mechanism(s) of action(s). Method: We performed a global search for potential ML297 binding sites in the GIRK/GIRKFD channel, and probed ML297 binding in channels by molecular Dynamics (MD) simulations. Furthermore, we examined ML297 effects on GIRK1/2 and GIRK1/4 heteromer channel expressed in Xenopus laevis oocytes by voltage clamp experiments. Results and Conclusion: In oocyte expression experiments, ML297 markedly stimulated GIRK1/GIRK4 channels similarly to GIRK1/GIRK2 channels. Strengthening channel-PIP2 interactions could occlude the ML297 effect in FD containing subunits of the channel. In silico docking and MD simulations suggest the potential binding site ML297 is located between the M1 and M2 helices, above the interfacial helix region, which may perturb the pore region. Moreover, ML297 binding in this region could affect channel-PIP2 interactions. 1249-Pos Board B317 Regulation of a Fungal K2P Channel by Protein Kinases Ryan Manville1, Andrew Corran2, Anthony Lewis1. 1 School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom, 2Syngenta, Bracknell, United Kingdom. Two-pore Outwardly-rectifying Kþ (TOK) channels are a family of structurally and functionally unique fungal K2P 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 cellular regulation by protein

kinases, which are known to be essential for fungal cell viability. Here, using two-electrode voltage clamp of Xenopus laevis oocytes expressing MgTOK channels, cloned from the wheat pathogen Zymoseptoria tritici, we study the impact of regulating protein kinase activity on MgTOK function. MgTOK currents were largely insensitive to the tyrosine kinase activator genestein (100 mM) but were increased by the PKA activator forskolin (100 mM, 1.0 5 0.1-fold at 0 mV, p = 0.003). PKC activation by 100 nM tetradecanoylphorbol acetate (PMA) also led to robust MgTOK current activation (3.8 5 0.3-fold at 0 mV, p = 0.0006) an effect that was concentration dependent (EC50 = 30.4 5 0.28 nM). The related phorbol ester, phorbol 12, 13-dibutyrate (PDB, 1 mM) similarly increased MgTOK currents at 0 mV (1.2 5 0.3-fold, p = 0.0003). PMA augmentation of MgTOK currents was almost completely inhibited by 1 mM bisindolylmaleimide II (Bis II), suggesting the involvement of cPKC isoform bII. Interestingly, application of PKA inhibitors fluoro-2-indolyl des-chlorohalopemide (KT5720, 2.5 mM) and protein kinase inhibitor peptide (PKI, 2.5 mM) resulted in a significant reduction in the positive effect of PMA, suggesting PMA may also activate PKA. Alanine and aspartate mutagenesis identified MgTOK-T344 as the major site of PKC phosphorylation, and MgTOK-S586 as a key site in PKA phosphorylation. Taken together, the regulation of MgTOK by protein kinases represents a potential target for future phytotherapeutic strategies. 1250-Pos Board B318 Silver as a Probe of Selectivity Filter Gating in TREK-1 K2P Channels Marcus Schewe, Thomas Baukrowitz. Institute of Physiology, Christian-Albrechts-Univerity of Kiel, Kiel, Germany. TREK-1 K2P channels are members of the mammalian two-pore domain (K2P) Kþ channel family. Channel activity is modulated by various physicochemical stimuli including temperature, pH, membrane stretch, cellular lipids and clinically relevant drugs. Although these gating mechanisms and underlying sensory structures within the channel have been investigated for years, direct evidence for the localization of the primary gating structure e.g. the pHsensitive gate in TREK-1 is currently missing. Previously, we have investigated the status of the helix-bundle cross (intracellular pore entrance gate) in TREK-1 during channel gating. Our findings established that the bundle crossing is constitutively open and suggested that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter (SF). To test this possibility directly, we here substituted residues around the selectivity filter signature sequence (GFG) by cysteines and probed their state dependence to modification by silver ions, which are known to permeate through the SF. We identified cysteines intracellular and extracellular near to the GFG that could be modified by silver ions applied from the intracellular side. Cysteines intracellular to the GFG were modified in the open and closed state, consistent with the intracellular access pathway to the SF being constitutively open and not gated. In contrast, cysteines extracellular to the GFG could be readily modified in active TREK-1 channels but were inaccessible to modification in closed channels, suggesting that the SF has entered a non-conductive state causing channel closure. Modification of the extracellular cysteines was also prevented when the pore was blocked by classical pore blockers (e.g. QAþ ions) and occurred only for currents passing in extracellular direction, ensuring that silver ions indeed moved through the SF to reach its extracellular modification site. These results unambiguously establish the SF as the primary gating structure in TREK-1 channels and promote silver modification as useful tool to probe selectivity filter gating regarding different regulatory stimuli in K2P channels. 1251-Pos Board B319 The Origin of Coupled Chloride and Proton Transport in a Cl-/HD Antiporter Sangyun Lee, Heather B. Mayes, Jessica M.J. Swanson, Gregory A. Voth. The University of Chicago, Chicago, IL, USA. ClC-ec1 is a bacterial Cl-/Hþ antiporter, which mediates the exchange mechanism of Cl- and Hþ ions through the membrane. It pumps either Cl- or Hþ in one direction through the membrane using a concentration gradient of the other ion in the opposite direction. Previous experimental studies revealed that E148 and E203 participate in proton transport (PT), and transport pathways for Cl- and Hþ in the protein overlap from E148 to the extracellular side of the protein, then diverge below E148. However, the underlying molecular mechanism of the Cl-/Hþ coupling has not been fully elucidated. Here we have developed the multiscale reactive MD model to explicitly simulate PT through water molecules and amino acids undergoing protonation and deprotonation reactions, and studied how much the energetics of PT from E203 to E148, and to the extracellular solution is affected by the presence of Cl- at the central site (Cl-cen). We found that E148 deprotonation is likely rate-determining for PT and facilitated by the presence of Cl-cen, however in the absence of Cl-cen, E148 is stabilized in the down conformation, effectively blocking PT from intra- to extracellular solution. Our results also confirmed the hypothesis