N-Linked Glycosylation Regulates CALHM1 Channel ... - Cell Press

Wednesday, March 2, 2016 shifted the voltage dependence of activation of SAKca to more negative voltages, whereas a relatively low concentration of extracellularly applied GsMTx-4 could reverse this effect. Detailed single-channel kinetics analysis revealed that GsMTx-4 could antagonize the increases in the open-time duration induced by membrane stretch. In addition, we found that the sensitivity of SAKca channel towards GsMTx-4 effect was eliminated by the deletion of 59-amino-acid STREX (stress axis regulated exon) located between RCK1 and RCK2 domains in the C-terminus of SAKca channel, suggesting that STREX is the potential target site for GsMTx-4 action in SAKca channel. Our data establish that GsMTx-4 acts as a gating modifier on SAKca channel from chick heart, and may represent a common inhibition mechanism of GsMTx-4 against all types of mechanosensitive (MS) ion channels. Our finding also suggest SA channel is a potential target for curing atrial fibrillation by using GsMTx-4, thus may provide a new insight into the treatment of cardiac arrhythmia. 3006-Pos Board B383 N-Linked Glycosylation Regulates CALHM1 Channel Function and Subcellular Localization Akiyuki Taruno1, Hongxin Sun1, Makiko Kashio1, Yoshinori Marunaka1,2. 1 Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Department of Bio-Ionomics, Kyoto Prefectural University of Medicine, Kyoto, Japan. Calcium homeostasis modulator 1 (CALHM1) was discovered as a susceptibility gene for late-onset Alzheimer’s disease. CALHM1 has been established as a pore-forming subunit of a homohexameric voltage-gated, non-selective ion channel and its important physiological roles including neuronal excitability and taste perception have also been revealed. However, other modes of CALHM1 channel regulation including ones mediated by post-translational modifications remain to be elucidated. On a western blot, heterologously expressed mouse CALHM1 (mCALHM1) immunosignal appears as three bands with one band at the size of a mCALHM1 monomer, approximately 37 kDa, and the other two bands at higher molecular weights, suggesting posttranslational modifications on CALHM1. The two higher molecular weight bands disppeared after peptide-N-glycosidase F treatment, demonstrating that mCALHM1 is glycosylated at asparagine (N) residue(s). Point mutation studies determined that a conserved N139 is the only N-glycosylation site on mCALHM1. The two glycosylated forms of mCALHM1 showed different sinsitivities to endoglycosidase H, demonstrating that conversion of the acquired N-linked glycan chain from high-mannose type to complex type takes place. Furthermore, by using chemical and enzymatic reagents, point mutation studies, and glycosylation mutant cell lines, we examined roles of acquisition and conversion of the N-linked glycan on function and subcellular localization of mCALHM1 channel. Our data provide insights into a novel regulation of CALHM1 channel by N-linked glycosylation. 3007-Pos Board B384 Effects of Excluded Volume and Induced N-Terminal Conformational Change on Ion Translocation across VDAC Sai Shashank Chavali, Grace Brannigan, Reza Salari. Center for Computational and Integrative Biology, Rutgers, The State University of New Jersey, Camden, NJ, USA. The Voltage Dependent Anion Channel (VDAC) is a mitochondrial outer membrane protein that mediates transfer of ions and small metabolites. It also allows apoptotic factors like cytochrome C into the cytoplasm, thereby playing a crucial role in mediating programmed cell death (apoptosis). Previous studies have indicated that Nicotinamide adenine dinucleotide in its reduced form (NADH) but not its oxidized form (NADþ) reduces conduction through VDAC. However, there is no available non-conducting structure of VDAC, and the mechanism of modulation by NADH remains poorly understood. Here we use long, fully-atomistic molecular dynamics simulations to study NADH binding to VDAC and its effect on channel dynamics and ion translocation. Simulations of VDAC were conducted using an NMR structure determined in the presence of NADH (and provided by Dr. Sebastian Hiller), and with NADH bound as in that structure, deprotonated in silico to NADþ, or removed entirely. We observed dissociation of NADþ within 100 ns, while NADH remained bound, suggesting that insensitivity of VDAC to NADþ reflects a significantly lowered affinity of NADþ for the VDAC pore. In VDAC with NADH entirely removed from the complex before simulation, the N-terminal loop dramatically changed its conformation over the course of the simulations, eventually approaching its conformation in structures experimentally determined in an apo-state. The present results are consistent with a mechanism in which NADH reduces conduction by partial pore block, while concurrently forcing a conformational change of the N-terminus. Potential direct contributions of the N-terminal loop to modulating conduction, such as reduction of the favorable ion density in the pore, are also discussed.

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3008-Pos Board B385 Electrophysiological Characterization of two Novel Ion Channels of Mitochondria Vanessa Checchetto1, Angela Paggio2, Simona Reina3, Diego De Stefani2, Vito De Pinto3, Rosario Rizzuto2, Ildiko` Szabo`1. 1 Department of Biology, University of Padua, Padua, Italy, 2Department of Biomedical Sciences, University of Padua, Padua, Italy, 3Department of Biological, Geological and Environmental Sciences, University of Catania, Catania, Italy. Mitochondrial ion channels are of great importance to ensure the proper function of this bioenergetic organelle and to regulate cell fate. However, in many cases our knowledge concerning their molecular identity and regulation is still limited. Here we describe a novel regulatory mechanism of the voltagedependent anion channel VDAC3 and a new channel-forming protein. Voltage-dependent anion channels (VDAC), also known as eukaryotic porins, are located in the outer mitochondrial membrane and allow the flux of ions and small metabolites. While the pore-forming ability of recombinant VDAC1 and VDAC2 has been extensively studied during the last decades, a clear-cut ion conducting channel activity has been assigned to the VDAC3 isoform only recently (Checchetto et al, Cell Phys Biochem, 2014). This protein forms an ion channel with small conductance under standard conditions, but our study identifies the amino acid residues whose oxidation state impacts on channel activity and conductance. Furthermore, we characterize from electrophysiological point of view a novel protein complex composed by pore-forming core and regulatory subunits located in the inner mitochondrial membrane. Channel activity can be observed with the reconstituted recombinant core protein, but for its regulation the co-expression of both the core and the regulatory subunits are necessary. Overall, our data suggest that VDAC3 and the novel protein complex are new players in the control of mitochondrial ion homeostasis and might contribute to the plasticity of mitochondrial function in intact cells. 3009-Pos Board B386 Conformational Changes that Opens TrkH Ion Channel Hanzhi Zhang, Zhao Wang, Wah Chiu, Ming Zhou. Baylor College of Medicine, Houston, TX, USA. All living cells must accumulate a high intracellular concentration of Kþ to maintain essential physiological functions. While in animals, Kþ uptake is mediated by the well-studied Naþ/Kþ transporters, bacteria, yeast and plants accumulate Kþ with a superfamily of Kþ transporters known as the SKT proteins. TrkH is a member of the SKT family that assembles with a cytosolic domain, TrkA. Previously, we have shown that TrkH is an ion channel regulated by binding of ATP or ADP to the TrkA protein: ATP activates the channel while ADP inhibits it (1). We have also solved the crystal structures of TrkH and TrkH in complex with TrkA (1, 2). The structure of TrkH shows that it forms a homodimer and that each protomer has an ion conduction pore with an architecture similar to that of a Kþ channel. The structure also revealed a feature not observed in Kþ channels: a loop within the transmembrane region that blocks the ion permeation pathway. Further structural and functional analyses showed that the position of this loop is likely controlled by conformational changes on TrkA, which is in turn controlled by binding of ATP or ADP. We have applied single-particle cryo-electron microscopy to visualize conformational changes associated with channel gating. Preliminary data show that the TrkH-TrkA complex likely assumes a different conformation than that observed in the crystal structure. 1. Cao, Y. et al. 2013. Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature 496:317-322. 2. Cao, Y. et al. 2011. Crystal structure of a potassium ion transporter, TrkH. Nature 471:336-340. 3010-Pos Board B387 Elucidating the pH Dependent Mechanism of OmpG Gating Christina M. Chisholm, Emily Friis, Monifa A. Fahie, Min Chen. Department of Chemistry, University of Massachusetts Amherst, Amherst, MA, USA. Protein pores are steadily emerging as valuable stochastic sensors for singlemolecule detection. Over the decade, developments in stochastic sensing have been based on single channel recording in planar lipid bilayer. Historically, nanopores with rigid structures have been frequently used for sensing molecules and biological species. However, we have chosen a nanopore with a flexible structure for our protein nanopore: monomeric b-barrel porin outer membrane protein G (OmpG). This monomeric porin is composed of 14- ßstrands connected by seven flexible extracellular loops. Wild-type OmpG spontaneously gates during an applied potential as revealed by planar bilayer studies. Strong evidence suggests that the dynamic movement of OmpG’s loop 6 is responsible for the gating mechanism of the porin. The unusual