Feb 13, 2017 - free-living organisms throughout evolutionary history. Specific ... we measured binding affinity of WT-CaM and two CPVT-linked CaM mutants.
Monday, February 13, 2017 from previous experimental studies about the transport mechanism. As a control, we have also simulated PT in the E148A mutant of ClC-ec1, where a nonprotonatable residue greatly increases the free energy barrier for PT from E203 to the extracellular solution, explaining the experimental result that PT in E148A is blocked whether or not Cl-cen is present. 1252-Pos Board B320 Phylogenetic and Sequence Analysis of the Flucs, a Remarkable Family of Fluoride Channels Christian B. Macdonald1, Randy B. Stockbridge2. 1 Biophysics Program, University of Michigan, Ann Arbor, MI, USA, 2 Biophysics Program and Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA. Fluoride, ubiquitous in the environment, has presented a persistent challenge to free-living organisms throughout evolutionary history. Specific mechanisms for dealing with this threat have recently been discovered, most notably the Flucs, a family of highly-selective fluoride channels. The Flucs are found in all kingdoms and offer a remarkable biophysical laboratory for investigating membrane channel evolution. Flucs assemble in the membrane as 4-transmembrane helix antiparallel dimers, with a two-fold symmetry axis parallel to the bilayer plane. Presumably arising from primordial gene duplication, examples have also been found for heterodimer and fused-domain Flucs in nature. Intriguingly, this pattern of internal duplication has been observed in transporters, with possible functional implications, but is much rarer in channels. Here we present a comprehensive phylogenetic and sequence analysis of crosskingdom Fluc channels. We observe evidence of multiple gene duplication events through clustering of clades. We examine the interplay of transmembrane helix hydrophobicity and loop charge bias (through the ‘‘positive-inside rule’’) in Fluc evolution. Further functional characterization of Fluc dimer symmetry through solution NMR is also shown. 1253-Pos Board B321 Mg2D Inhibits Cardiac SR Calcium Release and has Biphasic Effects on Calmodulin Binding to RyR2 Xiaoqiong Dong1, Ivanita Stefanon1,2, Rogerio F. Ribeiro Jr,1,2, Mena Said3, Robyn T. Rebbeck4, Razvan L. Cornea4, Donald M. Bers1. 1 Department of Pharmacology, University of California, Davis, Davis, CA, USA, 2Department of Physiological Sciences, Federal University of Espirito Santo, Victoria, Brazil, 3School of Medicine, University of California, Davis, Davis, CA, USA, 4Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA. Mg2þ is a potent inhibitor of RyR channel gating, as demonstrated in lipid bilayer and SR vesicles. Less is known about Mg2þ effects on RyR2 function in the cardiac myocyte environment or how it might alter RyR2 interaction with accessory proteins like calmodulin (CaM), which by itself binds to, and inhibits RyR2 opening. We used permeabilized mouse ventricular cardiomyocytes to study the effects of Mg2þ on spontaneous Ca2þ release and CaM affinity for RyR2. We recorded Ca sparks at three [Mg2þ]: 0.1 mM (low), 1 mM (physiological), and 3 mM (high). We found that in the presence of endogenous WT-CaM, with increasing [Mg2þ], Ca spark frequency (CaSpF) decreased monotonically, and was accompanied by an increase in Ca spark amplitude and SR Ca2þ load. To test whether Mg2þ also modulates RyR2 interaction with accessory proteins, we measured binding affinity of WT-CaM and two CPVT-linked CaM mutants (N54I and N98S). The apparent affinity of WT-CaM for RyR2 in myocytes was highest at 1 mM Mg2þ and decreased at both 0.1 mM and 3 mM Mg2þ. The same pattern was found for N54I- and N98S-CaM. We conclude that in cardiomyocytes, Mg2þ decreases CaSpF and increases SR Ca2þ load. As [Mg2þ] increases in the physiologically range (0.1 - 1 mM), part of the Ca spark inhibition may be due to enhanced CaM binding to RyR2. In contrast, pathophysiologically high [Mg2þ] (3 mM) may (1) limit RyR2-CaM binding (and thus CaM inhibition of RyR2) and (2) still directly inhibit RyR2 opening. 1254-Pos Board B322 Interactions between Neuronal Junctophilins and Voltage Gated Ion Channels Stefano Perni, Kurt G. Beam. Physiology and Biophysics, University of Colorado, Aurora, CO, USA. Junctions between the endoplasmic reticulum (ER) and plasma membrane (PM) occur in diverse cell types. In many of these junctions, calcium efflux from the ER occurs via ryanodine receptors or IP3 receptors, triggered by voltagegated ion channels or ligand receptors in the PM. Junctophilins (JPs) have been identified as proteins that cause formation of ER-PM junctions in muscle (JP1, JP2) and neurons (JP3, JP4). Knockout of JP1 or JP2 is perinatal or embryonic lethal. Single knockout of neuronal JP produces only a mild (JP3) or no (JP4) phenotype; knockout of both causes hippocampal abnormalities, irregular hindlimb reflexes and impaired memory. Here, we have examined the ability of neuronal
junctophilins to cause voltage-gated channels to localize within junctional domains of the PM by co-expressing, in tsA201 cells, voltage-gated channels and JP3 or JP4 tagged with different fluorescent proteins. In the absence of JPs, the L-type channel CaV1.2 had a relatively uniform distribution, while P/Q-type (CaV2.1), N-type (CaV2.2) and T-type (CaV3.1) channels were present in the surface at a low density making them difficult to visualize. Both JP3 and JP4 caused CaV1.2, CaV2.1 and CaV2.2 to cluster at sites co-localized with the junctophilin. Such clusters were not observed for CaV3.1. KV2.1 clustered without junctophilin at sites previously shown to represent PM junctions with the ER. When coexpressed, JP4 co-localized with the KV2.1 clusters, whereas JP3 did not, raising the possibility that the two junctophilins have distinct roles. Overall our results show that junctophilins are not just structural proteins necessary for ER-PM formation, but that they actively recruit neuronal channels to spatially restricted PM domains. Supported by: NIH grants AR070298 to KGB and GM109888 to MM Tamkun (who provided GFP-KV2.1). HM Colecraft provided CaV3.1-YFP. 1255-Pos Board B323 Voltage Dependent Inhibition of Cx46 Hemichannels by Calcium Bernardo I. Pinto, Amaury Pupo, Isaac Garcia, Karel Mena-Ulecia, Agustin Martinez, Ramon Latorre, Carlos Gonzalez. Centro Interdisciplinario de Neurociencias, Valparaiso, Chile. Connexins (Cxs) are transmembrane proteins involved in the electrical coupling of cells, the release of signaling molecules, and cell proliferation among others. These proteins are expressed in almost every tissue of the human body and mutations in these proteins are related to several hereditary diseases. Cxs monomers oligomerize in hexamers, which traffic to the plasma membrane and form connexons or hemichannels that can act as plasma membrane channels or can travel to intercellular contact zones where they form intercellular channels known as gap junctions. The opening of the Cx hemichannels in the membrane is tightly regulated by Ca2þ and membrane voltage. This regulation prevents leakage of cellular content and its malfunction may lead to pathologic conditions. In this work we studied the reciprocal regulation of Cxs hemichannels by Ca2þ and voltage. We expressed Cx46 in Xenopus laevis oocytes, and using two electrode voltage clamp, analyzed the inhibition of currents by Ca2þ. We observed that Cxs inhibition by Ca2þ is voltage dependent. The apparent Ca2þ sensitivity is increased as the membrane voltage is made more negative. This change in the affinity can be explained by a stabilization of the closed state of the Cx channel by Ca2þ. We propose a model in which the calcium-bonded state prevents the opening of the hemichannel. The model allows us to determine the affinity of the closed state for calcium. In agreement with the model and suggesting a stabilization of a conformational change that narrows the pore, calcium inhibits the water flux trough Cx hemichannels. This work supports the idea that Ca2þ and voltage act synergistically to promote the closing of the pore in Cx channels. 1256-Pos Board B324 Distribution of Phosphorylated Connexin 43 in Gap Junctions: Implications for Endocytosis Rachel A. Margraf, Rachael M. Kells Andrews, Matthias M. Falk. Biological Sciences, Lehigh University, Bethlehem, PA, USA. Gap junctions (GJs) are transmembrane protein complexes that form channels between cells and permit passage of small molecules. These channels are composed of protein subunits called connexins (Cxs), the most well studied being Cx43. Clusters of GJ channels form arrays known as GJ plaques. Specific phosphorylation and ubiquitination events on the C-terminal tail of connexins decrease intercellular communication, leading to endocytosis into annular gap junctions (AGJs) and degradation. Previous studies have identified phosphorylation (p) of Cx43 on Serines (S) 255, 262, 279/282, and 368 as associated with decreased GJ intercellular communication (The´venin et al. 2013), but the significance and order of these events is not well understood. We hypothesize pS368 occurs early in the pathway, while pS279/282 and possibly pS255 and pS262 occur later. Because new GJ channels are added to the rim of the plaque, later phosphorylation events are expected to occur towards the center. Using antibodies specific to pS279/S282 and pS368, we examined their distribution within GJ plaques using fluorescence microscopy. We found pS279/S282 Cx43 located in GJ plaque centers, while pS368 Cx43 was present at the periphery and central plaque areas. These results suggest pS368 occurs earlier than pS279/S282, and these events aid in signaling GJ internalization. We are continuing to utilize phosphorylation specific antibodies to analyze the distribution of other Cx43 phosphorylations. We are also analyzing the phosphorylation distribution of mutant Cx43 that have Lysines exchanged for Arginines at possible ubiquitination sites. This will further elucidate the order of ubiquitination and phosphorylation events. If internalization cannot proceed without a specific ubiquitination, then GJs channels will accumulate in the phosphorylation state that occurs prior to ubiquitination. Our findings will contribute to deciphering on a molecular level how GJ endocytosis is initiated and regulated.