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Anion exchanger 1 (AE1, band 3) is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member.

Spontaneous Ca(2+) release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca(2+) release (SOICR) can result in Ca(2+) waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here we show that a point mutation, E4872A, in the helix bundle crossing region (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, Ca(2+) activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni(2+)-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca(2+)-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca(2+), explaining the regulation of RyR2 by luminal Ca(2+), the initiation of Ca(2+) waves and Ca(2+)-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.

Long chain fatty acids are biologically important molecules with complex and pH sensitive aggregation behavior. The carboxylic head group of oleic acid is ionizable, with the p[K.sub.a] shifting to larger values, even above a value of 7, in certain aggregate states. While experiments have determined the macroscopic phase behavior, we have yet to understand the molecular level details for this complex behavior. This level of detail is likely required to fully appreciate the role of fatty acids in biology and for nanoscale biotechnological and industrial applications. Here, we introduce the use of constant pH molecular dynamics (MD) simulations with the coarse-grained MARTINI model and apply the method to oleic acid aggregates and a model lipid bilayer. By running simulations at different constant pH values, we determined titration curves and the resulting p[K.sub.a] for oleic acid in different environments. The coarse-grained model predicts positive p[K.sub.a] shifts, with a shift from 4.8 in water to 6.5 in a small micelle, and 6.6 in a dioleoylphosphatidylcholine (DOPC) bilayer, similar to experimental estimates. The size of the micelles increased as the pH increased, and correlated with the fraction of deprotonated oleic acid. We show this combination of constant pH MD and the coarse-grained MARTINI model can be used to model pH-dependent surfactant phase behavior. This suggests a large number of potential new applications of large-scale MARTINI simulations in other biological systems with ionizable molecules. Key words: constant pH molecular dynamics, fatty acids, oleic acid, MARTINI model, coarse-grained model. Les acides gras a longue chaine sont des molecules importantes sur le plan biologique, dont le comportement d'agregation est complexe et sensible au pH. Le groupe carboxyle de tete de l'acide oleique est ionisable, le pKa evoluant vers des valeurs plus elevees, meme superieures a 7, dans certains etats agreges. Si des experiences ont permis de determiner le comportement dans la phase macroscopique, on n'a pas encore elucide les details de niveau moleculaire de ce comportement complexe. Ce niveau de detail est vraisemblablement necessaire pour apprecier pleinement le role des acides gras en biologie, ainsi que pour les applications biothechnologiques et industrielles nanoscopiques. Ici, nous presentons l'utilisation de simulations de dynamique moleculaire a pH constant a l'aide du modele gros-grain MARTINI et nous appliquons la methode aux agregats d'acide oleique et a un modele de bicouche lipidique. En executant les simulations a differents pH, nous determinons les courbes de titrage et le pKa resultant de l'acide oleique dans diverses conditions. Le modele gros-grain predit des changements positifs de pKa, lequel passe de 4,8 dans l'eau a 6,5 dans une petite micelle, et a 6,6 dans une bicouche de DOPC, qui sont similaires aux estimations experimentales. La taille des micelles s'accroft quand le pH augmente et correle avec la fraction d'acide oleique deprotone. Nous montrons que l'on peut se servir de cette combinaison de dynamique moleculaire a pH constant et du modele de simulation gros-grain MARTINI pour modeliser le comportement variant avec le pH dans la phase tensioactive. Cela suggere un grand nombre de nouvelles applications possibles de simulations MARTINI a grande echelle pour d'autres systemes biologiques comprenant des molecules ionisables. [Traduit par la Redaction] Mots-cles: dynamique moleculaire a pH constant, acides gras, acide oleique, modele Martini, modele gros-grain., Introduction Surfactants are crucial chemicals in our daily lives, from soaps and detergents to food processing and biotechnology. Fatty acid aggregation is important for a wide range of applications. Fatty [...]