during development. The improvement coincides with the selective disappear- ance of the slowest component of synaptic decay. We recorded single channel properties of heterologously expressed zebrafish subunits a twi along with b,d,e or g subunits to identify the basis of this slowest component. a twi bde receptors exhibit a mean open time that corresponds to the intermediate time constant of twister decay. By contrast, the embryonic receptor isoform a twi bdg exhibited a greatly prolonged open time that corresponded to the slowest component of synaptic decay in twister. This result is unexpected given the similarity in mean open times for wild type abde (0.5 ms) and abdg (1.2 ms) receptors. We propose that behavioral and functional recovery results from the observed developmental decrease in g subunit mRNA expression, facilitating a switch from a twi bdg to a twi bde receptors. In support of this idea, morpholino RNA knockdown of g subunit expression improved swimming performance and re- duced the contribution of the slow component to synaptic current decay, pre- sumably through an increase in a twi bde AChRs. 483-Pos Board B283 Kinetic Control of SNARE-Dependent Fusion by Accessory Factors and Calcium Minjoung Kyoung, Ankita Srivastava, Yunxiang Zhang, Marija Vrljic, Patricia Grob, Eva Nogales, Steven Chu. Ca 2þ triggers neurotransmitter release to initiate signal transmission in all syn- apses. Synaptotagmin 1 is the Ca 2þ -sensor for fast synchronous release, com- plexin is a modulator, and SNAREs are essential for synaptic vesicle fusion. Yet, the molecular mechanism of Ca 2þ -triggering remains unclear. Here we de- scribe the first successful reconstitution of this machinery at a single vesicle level that captures some of the kinetic and cooperative properties observed in vivo. Starting from a defined state of docked vesicles, simultaneous content and lipid mixing indicators differentiate between hemifusion and fusion. Upon Ca 2þ -injection half of the docked donor vesicles fuse with acceptor ves- icles within 0.27 secs when functional SNAREs, synaptotagmin 1, and com- plexin are reconstituted. In contrast, we observe a much slower response with SNAREs alone, or non-functional mutants of synaptotagmin 1 or com- plexin. However, fusion occurrence over a long time period is unaffected by these mutants, suggesting that synaptotagmin 1 and complexin are Ca 2þ -con- trolled kinetic regulators of SNARE-induced fusion. Ion Channels, Other 484-Pos Board B284 Ion Channels, Action Potentials and Ca 2þ Handling in Human Pancreatic Beta-Cells. a Computational Approach Leonid E. Fridlyand, Louis H. Philipson. In this study, we examined the ionic mechanisms mediating depolarization- induced spike activity in human pancreatic beta-cells. We formulated a Hodg- kin-Huxley-type ionic model for the action potential (AP) in these cells based on experimental voltage- and current-clamp results from our laboratory and lit- erature. The model contains the equations for the currents: inward L-, P/Q and T-type Ca 2þ , a ‘‘rapid’’ delayed rectifier K þ , the voltage-gated and Ca 2þ -acti- vated K þ (BK-type), a voltage-independent Ca 2þ -activated K þ (SK-type), an ATP-sensitive K þ , a plasma membrane calcium pump, a voltage-gated Na þ and a Na þ background. Ionic model is coupled to an equation describing intra- cellular Ca 2þ homeostasis. The model simulates the behavior of human beta- cell APs under a wide range of experimental conditions, including changes in the period and amplitude of AP in response to glucose challenge and changes in islet electrical activity due to K þ , Na þ and Ca þ channel blockade. The model was used to study the role of specific ionic currents in human pancreatic beta- cell firing. Particularly, modeling supports the importance of constitutively ac- tive tetrodotoxin-sensitive Na þ and voltage-gated Ca 2þ -activated K þ channels (BK-type) in maintaining spontaneous spikes. This model provides acceptable fits to voltage-clamp, action potential and Ca 2þ concentration data and can be used to seek biophysically based explanations of the electrophysiological activ- ity and Ca 2þ influx in human beta-cells for in silico analysis of physiological conditions and channel modulator actions. 485-Pos Board B285 Theoretical Biophysics of Ion Concentration Effects on the Affinity of Nanopore OmpF Lumen to Nucleotides Saeid Hadi Alijanvand, Hamid Mobasheri. The OmpF porin from the Escherichia coli outer membrane folds into a tri- mer of ß -barrels, each forming an aqueous pore allowing the passage of ions and small solutes. One of external loops, long L3 carrying multiple acidic residues folds into the ß -barrel pore to form a narrow ‘‘constriction zone’’ in the mid height of the channel. The intrinsic charge distribution in the conducting path may be affected by many factors. Nucleotides are known as one of the most important metabolites in the cell that can pass through membrane when they are needed. The experimental findings on translocation of such molecules through channels have provided useful infor- mation. Considering instrumental limitations , theoretical studies , modelings and simulations are applied. In this study we calculated the changes in the affinity of OmpF nanochannel lumen to pyrimidin and purine bases at mo- lecular level by Delphi 4. A non-linear Poisson-Boltzmann calculator with definite element integrator found to be appropriate choice to calculate the net energy of OmpF-nucleotide interactions at different ion concentration. Our results indicate that decreasing in the ion concentration led to increase in the OmpF affinity to purine and pyrimidine bases. The calculation outputs indicated that OmpF- affinity for pyrimidine is higher than that of purine. These observations may shed light to understand the selective molecular translocation means implemented by OmpF. 486-Pos Board B286 A novel Gene Required for Male Fertility and Functional Catsper Channel Formation in Spermatozoa Jean-Ju L. Chung, Betsy Navarro, Grigory Krapivinsky, Luba Krapivinsky, David E. Clapham. Calcium (Ca2þ) signaling is critical for successful fertilization. In spermato- zoa, capacitation, hyperactivation of motility, and the acrosome reaction are mediated by changes in the intracellular Ca2þ. Cation channels of sperm (CatSpers1-4) are alkalinization-activated Ca2þ-selective ion channels con- trolling hyperactivated motility of spermatozoa and male fertility. CatSpers form a heteromeric complex with each of the CatSpers1-4 surrounding a Ca2þ-selective pore, in analogy with other six-transmembrane (6TM) ion channel alpha subunits. In addition to the pore-forming proteins, the sperm Ca2þ channel contains auxiliary subunits, CatSper beta and CatSper gamma. Here, we identify the Tmem146 gene product as another novel CatSper acces- sory subunit, CatSper delta. We find that mice lacking the sperm tail-specific CatSper delta are infertile and their spermatozoa lack both CatSper current and hyperactivated motility. We show that CatSper delta has a central role in CatSper channel complex stability during spermatogenesis, providing new in- sight into the function of auxiliary subunits and the mechanism of CatSper channel complex assembly. 487-Pos Board B287 Self-Assembly of a Small Synthetic Molecule forms Well-Defined Ion Channels Panchika Prangkio, Divya Rao, Mark Rubinshtein, Jerry Yang, Michael Mayer. Synthetic ion channels are interesting for pharmacological and biomedical ap- plications, including the development of antimicrobial agents, drug delivery ve- hicles, and biosensors. Previous work demonstrated that the attachment of polyethyleneglycol (PEG) groups to a molecular template such as cyclodextrin can lead to the formation of pore-forming molecules in lipid membranes. Here, we introduce small synthetic molecules based on oligo(ethylene glycol) and benzothiazol ani- line (BTA) deriva- tives, which self- assemble in lipid bilayer to form ion channels with very-well defined ion conductances. Remarkably, these self-assembled ion pores were selective for monovalent cations and could be ‘‘gated’’ by changes in pH. 488-Pos Board B288 Energetics and Molecular Mechanisms of Permeation and Selectivity of Transport in the Urea Transporter Giray Enkavi, Elena J. Levin, Ming Zhou, Emad Tajkhorshid. Urea, ubiquitously used as a nitrogen source by bacteria and a safe end prod- uct of protein catabolism, depends on a specialized facilitator, urea trans- porter (UT) for its selective transport across the plasma membrane. Despite the name ‘‘transporter’’, UT has been suggested to operate by a chan- nel like mechanism owing to its high transport rate. The crystal structure of a bacterial UT was reported recently as a homotrimer in the apo and sub- strate (dimethylurea; DMU)-bound states. However, important transport characteristics, such as urea binding sites and water permeability, have not been identified. To understand these transport properties of UT, we have per- formed extended equilibrium molecular dynamics simulations of the UT tri- mer with each monomer in one of the apo, DMU-bound, and urea-bound (modeled by Ming Zhou group) states, as well as umbrella sampling simu- lations of urea. These simulations allowed us to characterize expulsion 90a Sunday, March 6, 2011