Sumoylation of NaV1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

Monday, February 13, 2017 properties of the brain GIRK2 channel in a defined reconstituted system that allowed for precise control of small molecules and ions. We found that cholesterol, a major component of brain membranes, potentiates the neuronal GIRK2 channel activity in a PIP2 dependent manner. Cholesterol shifts the PIP2 dose-response curve to lower concentrations, demonstrating enhanced PIP2 binding to the channel. Ethanol activates GIRK channels independently of the agonist-mediated pathway via binding to an alcohol pocket in the cytoplasmic terminal domain. There is no apparent shift in alcohol sensitivity in the presence of cholesterol, suggesting alcohol and cholesterol interact structurally and mechanistically with different regions of the channel. Elucidating the molecular mechanism underlying the activation of GIRK channels by small compounds, such as ethanol and cholesterol, should aid in the design of new drugs for treating alcoholism and other neurological disorders. 902-Plat BK Channel a and g1 Subunits Assemble with a Stoichiometry of up to 4:4, but One g1 is Sufficient to Produce the Full g1-Induced Gating Shift Vivian Gonzalez-Perez1, Manu Ben-Johny2, Xiao-Ming Xia1, Christopher J. Lingle1. 1 Anesthesiology, Washington University in St. Louis, St. Louis, MO, USA, 2 Biomedical Engineering, The Johns Hopskins University School of Medicine, Baltimore, MD, USA. Ca2þ-and-voltage-gated Kþ-channels of large conductance (BK) are symmetrical complexes whose minimal functional unit is a tetramer composed of four identical a-subunits. BK a-subunits can coassemble with auxiliary subunits belonging to two different families, b and g, which drastically influence channel gating. The presence of b2 subunits produces a complete inactivation of BK currents and the inactivation rate at single channel level is a functional reporter of the number of b2 subunits/channel. This methodology has established that up to four b-subunits can assemble with the a-subunits in a BK channel complex, where each one contributes an identical additive increment to the total b-induced gating shift. However, the stoichiometry of the assembly of a and g subunits is still unknown. In order to solve it, we designed an ‘‘inactivating’’ g1 subunit (chimera b2/g1) and measured the inactivation rates at the single channel level. Our results show that, similar to b subunits, up to four b2/g1 chimeric subunits can assemble in BK channels expressed in Xenopus oocytes. Unlike b subunits, the presence of one b2/g1 chimeric subunit in a single BK channel is sufficient to produce the full g1-induced gating shift. Our results confirm the ‘all-or-none’ type of functional regulation produced by g1 on BK channels. Independent FRET-based methods also confirm that up to four tagged g1 subunits can be present per BK channel (Supported by GM118114 to CJL). 903-Plat Sumoylation of NaV1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons Leigh D. Plant1, Jeremy D. Marks2, Steve A.N. Goldstein1. 1 Biochemistry, Brandeis University, Waltham, MA, USA, 2Pediatrics, University of Chicago, Chicago, IL, USA. Acute hypoxia is a common, dangerous, clinical condition that can disrupt electrical signaling in the brain and, in the presence of substrate deprivation, can initiate the death of neurons. At the cellular level, hypoxia triggers a neurotoxic cascade that begins with a rapid increase in sodium flux (INa). The basis for acute hypoxia-induced augmentation of INa has been unknown. Here, we combine electrophysiology, single molecule spectroscopy (TIRF) and Fo¨rster Resonance Energy Transfer (FRET) to show that hypoxia activates the Small Ubiquitin-like Modifier (SUMO) pathway in rat cerebellar granule neurons and that SUMOylation of NaV1.2 channels increases INa. The time-course for SUMOylation of single NaV1.2 channels at the cell surface and changes in INa coincide, and both are prevented by mutation of a single site in the NaV1.2 a-subunit or application of a deSUMOylating enzyme. Within 40 s, hypoxia-induced linkage of SUMO1 to the channels is complete, shifting the half-activation curve by up to 26.5 mV so that voltage steps evoke larger Naþ currents. Given the recognized role of INa in hypoxic brain damage, the SUMO pathway and NaV1.2 are identified as potential targets for neuroprotective interventions. 904-Plat Stoichiometries of HCN-KCNE2 Channel Yoann Lussier, Lena Mo¨ller, Oliver F€urst, Rikard Blunck, Nazzareno D’Avanzo. University of Montreal, Montreal, QC, Canada. HCN channels play crucial roles in regulating heart beats and neuronal firing. The KCNE2 auxiliary subunit acts as an important regulator of HCN channel function, with different effects on each HCN isoform. Here we explore the stoichiometries of HCN channel pore-forming subunits with the KCNE2 subunit

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using a single-molecule fluorescence photo bleaching technique. We also examine the implications that KCNE2 mutations linked to sudden death have on these protein-protein interactions. 905-Plat Molecular Interactions between Kv4.3 and DPP6 - The Biochemical Anatomy of Idiopathic Ventricular Fibrillation Nazlee Sharmin1, Peter Mohler2, Rikard Blunck3, Stanley Nattel1. 1 Pharmacology and Therapeutics, McGill University and Montreal Heart Institute, Montreal, QC, Canada, 2Ohio State University, Columbus, OH, USA, 3Universite´ de Montre´al, Montreal, QC, Canada. The molecular basis of idiopathic ventricular fibrillation (IVF) is poorly understood. Previously, we identified crucial role of DPP6, a Kv4.3associating b-subunit, in Purkinje fiber Ito and gene variants causing DPP6 gain-of-function in IVF. This study investigated the molecular basis of Kv4.3-DPP6 interaction, about which little is known. A computational model of the Kv4.3-DPP6 interaction (using AutoDock Vina) predicted that the N-terminus and some charged regions of DPP6 might interact with the N-terminus and 3 positively charged residues of Kv4.3. The predictions were verified in a patch-clamp study of wildtype and engineered mutant proteins expressed in HEK cells. Wildtype Kv4.3 showed substantial current enhancement upon co-expression with DPP6 after 48 hrs, as previously reported. Deletion of 5 amino acids in the Kv4.3 N-terminus decreased the peak-current density compared to wildtype Kv4.3 but did not prevent current-enhancement by wildtype DPP6, suggesting that this region of Kv4.3 N-terminus is not essential for interaction with DPP6. Neutralization of the 3 positively charged amino acids in the Kv4.3 N-terminus by alanine mutation enhanced the DPP6-induced current-increase. On the other side, deletion of the N-terminal region of DPP6 prevented its current-enhancing effect on Kv4.3. Neutralization of either positively or negatively charged regions in DPP6 significantly attenuated its ability to increase current density when co-expressed with wildtype Kv4.3. We conclude that Kv4.3 interacts with DPP6 via docking involving the DPP6 N-terminus, and that crucial charged amino acids on both Kv4.3 and DPP6 strongly affect the interaction. However, the opposite effects of charge neutralization in the Kv4.3 and DPP6 N-termini argue against a simple electrostatic interaction. These results provide insights into the biochemical anatomy of IVF and may help to develop effective molecularly-targeted blockers for patients with IVF-inducing DPP6 gain-of-function. 906-Plat Characterization of the Orai-Calmodulin Interaction as Potential Mediator of Calcium-Dependent Orai-Channel Inactivation Lukas Traxler1, Felix Faschinger1, Petr Rathner2, Michael Stadlbauer1, Tatsiana Charnavets3, Christoph Romanin1, Norbert M€uller2, Peter Hinterdorfer1, Hermann J. Gruber1. 1 Institute of Biophysics, Johannes Kepler University, Linz, Austria, 2Institute of Organic Chemistry, Johannes Kepler University, Linz, Austria, 3CF Centre of Molecular Structure, BIOCEV, Vestec, Czech Republic. Calcium is an important second messenger involved in many cellular processes such as cell proliferation, T-cell activation, muscle contraction, egg fertilization, or apoptosis. Calcium entry into non-excitable cells is mainly carried by store-operated Ca2þ release-activated Ca2þ (CRAC) channels, whereby Orai was found to be the calcium channel in the plasma membrane and the stromal interaction molecule (STIM) to act as a calcium sensor in the endoplasmatic reticulum (ER), and as an activator of Orai channels. A reduction of Ca2þ in the ER causes STIM to oligomerize, enabling its accumulation at the ER-plasma membrane junctions where it binds directly to Orai to open the Ca2þ-channel. Compared with activation, much less is known about the mechanisms underlying Ca2þ-dependent inactivation (CDI) processes. It has been proposed that binding of calmodulin (CaM) to a highly conserved N-terminal segment of Orai1 adjacent to its first transmembrane helix is critical for CDI. Hereby it is assumed that CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation. However, the molecular basis of these interactions remains unclear. In this study, the interaction between calmodulin and the N-terminal fragment of Orai1 and its homologue Orai3 was studied by Single Molecule Recognition Force Spectroscopy, Surface Plasmon Resonance, and Microscale Thermophoresis. Both Orai fragments showed a calcium-dependent and highly specific interaction with calmodulin, with slightly longer bond lifetimes and lower dissociation rate constants for Orai3 than for Orai1. Our data support a switch between a 1:1 and a 1:2 binding stoichiometry between CaM and Orai1/3. The latter occurs at low CaM concentration, has much higher affinity, and takes much longer to disscociate after removal of Ca2þ by EGTA. By comparison, only 1:1 binding with high affinity was found between CaM and smooth muscle