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Na + /Ca 2+ exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca 2+ homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na + or Ca 2+ , and thus can catalyze uphill Ca 2+ transport driven by a Na + gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na + ions occupy the so-called S ext , S Ca , and S int sites, whereas the S mid site is occupied by one water molecule and one H + , which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na + /Ca 2+ and Ca 2+ /Ca 2+ exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na + and Ca 2+ requires protonation of D240, and that this side chain does not coordinate either ion at S mid . These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na + across the membrane is electrogenic, whereas transport of Ca 2+ is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.secondary transporters | membrane antiporters | ion specificity | CaCA superfamily | molecular-dynamics simulations C a 2+ signals control a variety of cellular processes essential for the basic function of multiple organs. In cardiac cells, for example, Ca 2+ release from the sarcoplasmic reticulum is a necessary step for heart contraction, whereas Ca 2+ extrusion from the cell is required for cardiac relaxation. These fluctuations in the cytosolic Ca 2+ concentration underlie the initiation of the heartbeat (1, 2). Na + /Ca 2+ exchangers (NCXs) play a central role in the homeostasis of cellular Ca 2+ (3-5). These integral membrane proteins are ubiquitous in many types of tissues including the heart, brain, and kidney (4), and consequently their dysfunction is associated with numerous human pathologies such as cardiac arrhythmia, hypertension, skeletal muscle dystrophy, and postischemic brain damage (5). NCXs facilitate the translocation of either Ca 2+ or Na + across the membrane; thus, they can harness a transmembrane sodium motive force to energize Ca 2+ transport against a concentration gradient. For example, the cardiac exchanger NCX1 mediates the extrusion of intracellular Ca 2+ driven by a Na + transmembrane gradient maintained by the Na ...

In analogy with many other proteins, Na+/Ca2+ exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na+/Ca2+ exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na+or Ca2+ binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct “noncatalytic” residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.

Na/Ca exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj ( NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na or Ca specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity ( ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

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Most modern compilers operate by applying a fixed, program-independent sequence of optimizations to all programs. Compiler writers choose a single "compilation sequence", or perhaps a couple of compilation sequences. In choosing a sequence, they may consider performance of benchmarks or other important codes. These sequences are intended as general-purpose tools, accessible through command-line flags such as -O2 and -O3.Specific compilation sequences make a significant difference in the quality of the generated code, whether compiling for speed, for space, or for other metrics. A single universal compilation sequence does not produce the best results over all programs [8, 10, 29, 32]. Finding an optimal program-specific compilation sequence is difficult because the space of potential sequences is huge and the interactions between optimizations are poorly understood. Moreover, there is no systematic exploration of the costs and benefits of searching for good (i.e., within a certain percentage of optimal) program-specific compilation sequences.In this paper, we perform a large experimental study of the space of compilation sequences over a set of known benchmarks, using our prototype adaptive compiler. Our goal is to characterize these spaces and to determine if it is cost-effective to construct custom compilation sequences. We report on five exhaustive enumerations which demonstrate that 80% of the local minima in the space are within 5 to 10% of the optimal solution. We describe three algorithms tailored to search such spaces and report on experiments that use these algorithms to find good compilation sequences. These experiments suggest that properties observed in the enumerations hold for larger search spaces and larger programs. Our findings indicate that for the cost of 200 to 4,550 compilations, we can find custom sequences that are 15 to 25% better than the human-designed fixed-sequence originally used in our compiler.

Voltage-dependent calcium channels (VDCCs) allow the passage of Ca 2ϩ ions through cellular membranes in response to membrane depolarization. The channel pore-forming subunit, ␣1, and a regulatory subunit (Ca V ␤) form a high affinity complex where Ca V ␤ binds to a ␣1 interacting domain in the intracellular linker between ␣1 membrane domains I and II (I-II linker). We determined crystal structures of Ca V ␤2 functional core in complex with the Ca V 1.2 and Ca V 2.2 I-II linkers to a resolution of 1.95 and 2.0 Å, respectively. Structural differences between the highly conserved linkers, important for coupling Ca V ␤ to the channel pore, guided mechanistic functional studies. Electrophysiological measurements point to the importance of differing linker structure in both Ca V 1 and 2 subtypes with mutations affecting both voltage-and calcium-dependent inactivation and voltage dependence of activation. These linker effects persist in the absence of Ca V ␤, pointing to the intrinsic role of the linker in VDCC function and suggesting that I-II linker structure can serve as a brake during inactivation.

Background: Calmodulin and calcium-binding protein 1 (CaBP1) oppositely regulate inactivation of Ca V 1.2 channels. Results: Quantitative titration of purified proteins in intact cells suggests competition between CaM and CaBP1 for the Ca V 1.2 C terminus and an additional non-competitive action of CaBP1. Conclusion: CaBP1 counteracts CaM actions by a dual mechanism. Significance: Our approach provides new insights into mechanisms of Ca 2ϩ channel inactivation.