The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from the cytosol into the sarco-endoplasmic reticulum (SR/ER) lumen. Calcium is an important signaling ion, and the activity of SERCA is critical for a variety of cellular processes such as muscle contraction, neuronal activity, and energy metabolism. SERCA is regulated by several small transmembrane peptide subunits that are collectively known as the “regulins”. Phospholamban (PLN) and sarcolipin (SLN) are the original and most extensively studied members of the regulin family. PLN and SLN inhibit the calcium transport properties of SERCA and they are required for the proper functioning of cardiac and skeletal muscles, respectively. Myoregulin (MLN), dwarf open reading frame (DWORF), endoregulin (ELN), and another-regulin (ALN) are newly discovered tissue-specific regulators of SERCA. Herein, we compare the functional properties of the regulin family of SERCA transmembrane peptide subunits and consider their regulatory mechanisms in the context of the physiological and pathophysiological roles of these peptides. We present new functional data for human MLN, ELN, and ALN, demonstrating that they are inhibitors of SERCA with distinct functional consequences. Molecular modeling and molecular dynamics simulations of SERCA in complex with the transmembrane domains of MLN and ALN provide insights into how differential binding to the so-called inhibitory groove of SERCA—formed by transmembrane helices M2, M6, and M9—can result in distinct functional outcomes.

The sarcoplasmic reticulum Ca-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.

Aciniform silk protein (AcSp1) is the primary component of wrapping silk, the toughest of the spider silks because of a combination of high tensile strength and extensibility. Argiope trifasciata AcSp1 contains a core repetitive domain with at least 14 homogeneous 200-amino acid units ("W" units). Upon fibrillogenesis, AcSp1 converts from an α-helix-rich soluble state to a mixed α-helical/β-sheet conformation. Solution-state nuclear magnetic resonance (NMR) spectroscopy allowed demonstration of variable local stability within the W unit, but comprehensive characterization was confounded by spectral overlap, which was exacerbated by decreased chemical shift dispersion upon denaturation. Here, (19)F NMR spectroscopy, in the context of a single W unit (W1), is applied to track changes in structure and dynamics. Four strategic positions in the W unit were mutated to tryptophan and biosynthetically labeled with 5-fluorotryptophan (5F-Trp). Simulated annealing-based structure calculations implied that these substitutions should be tolerated, while circular dichroism (CD) spectroscopy and (1)H-(15)N chemical shift displacements indicated minimal structural perturbation in W1 mutants. Fiber formation by W2 concatemers containing 5F-Trp substitutions in both W units demonstrated retention of functionality, a somewhat surprising finding in light of sequence conservation between species. Each 5F-Trp-labeled W1 exhibited a unique (19)F chemical shift, line width, longitudinal relaxation time constant (T1), and solvent isotope shift. Perturbation to (19)F chemical shift and nuclear spin relaxation parameters reflected changes in the conformation and dynamics at each 5F-Trp site upon addition of urea and dodecylphosphocholine (DPC). (19)F NMR spectroscopy allowed unambiguous localized tracking throughout titration with each perturbant, demonstrating distinct behavior for each perturbant not previously revealed by heteronuclear NMR experiments.

SUMMARY PARAGRAPHPhysiological systems require feedback to maintain normal function. In the heart, electrical excitation causes mechanical contraction1, with feedback of mechanics to electrics occurring through ‘mechano-electric coupling’ processes2. In diseases that affect cardiac mechanics, this feedback can result in deadly mechanically-induced arrythmias (‘mechano-arrhythmogenicity’)3. However, the molecular identity of the specific factor(s) driving mechano-arrhythmogenicity are unknown4. Here we show that mechano-sensitive5–10 transient receptor potential kinase ankyrin 1 (TRPA1) channels11 are a source of cardiac mechano-arrhythmogenicity through a calcium (Ca2+)-driven mechanism. Using a cell-level approach involving stretch of single ventricular myocytes combined with simultaneous voltage-Ca2+ imaging, we found that activation of TRPA1 channels resulted in an increase in diastolic Ca2+ load and the appearance of stretch-induced arrhythmias, which were driven by trans-sarcolemmal fluxes and intracellular oscillations of Ca2+, and prevented by pharmacological TRPA1 channel block or Ca2+ buffering. Our results demonstrate that TRPA1 channels act as a trigger for stretch-induced excitation (via Ca2+-influx) and create a substrate for complex arrhythmic activity (via Ca2+-overload), and thus may represent a novel anti-arrhythmic target in cardiac diseases in which TRPA1 channel expression and activity are augmented12–16.

From insects to humans, calcium signaling is essential for life. An important part of this process is the sarco-endoplasmic reticulum calcium pump SERCA, which maintains low cytosolic calcium required for intracellular calcium homeostasis. In higher organisms, this is a tightly controlled system where SERCA interacts with tissue-specific regulatory subunits such as phospholamban in cardiac muscle and sarcolipin in skeletal muscle. With the discovery of the sarcolambans, the family of calcium pump regulatory subunits also appears to be ancient, spanning more than 550 million years of evolutionary divergence from insects to humans. This divergence is reflected in the peptide sequences, which vary enormously from one another and range from vaguely phospholamban-like to sarcolipin-like. Our goal was to investigate sarcolamban peptides for their ability to regulate calcium pump activity. For a side-by-side comparison of diverse sarcolamban peptides, we tested them against mammalian skeletal muscle SERCA1a. This allowed us to determine if the sarcolamban peptides mimic phospholamban and sarcolipin in their regulatory activities. Four sarcolamban peptides were chosen from different invertebrate species. Of these, we were able to purify sarcolamban peptides from bumble bee, water flea, and tadpole shrimp. Sarcolamban peptides were co-reconstituted into proteoliposomes with SERCA1a and the effect of each peptide on the calcium affinity and maximal activity of SERCA was measured. While all peptides were super-inhibitors of SERCA, they exhibited either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations were used to reveal novel features of insect versus mammalian calcium pumps and the sarcolamban regulatory subunits.

Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein−protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.

Introduction: Clinical evidence indicates that in hypertension, acute fluctuations in blood pressure can trigger ventricular arrhythmias. Single cell rabbit studies suggest that these stretch-induced arrhythmias (SIA) are related to increased microtubule (MT) density or detyrosination and depend on transient receptor potential ankyrin 1 channels (TRPA1). However, whether these mechanisms of SIA apply to the whole heart is unknown. Hypothesis: An increase in MT density or detyrosination will reduce the threshold for SIA in the whole heart and be dependent on TRPA1. Methods: Isolated rabbit hearts were instrumented with surface electrodes to monitor electrical activity and an intraventricular balloon to alter LV load. Transient changes in LV volume were applied to induce SIA (50-500μL, 10mL/s, 20 repetitions of each volume separated by 30s). SIA incidence vs volume was used to calculate the volume that resulted in a 50% SIA incidence (V 50 ). Paclitaxel (5μM) was applied alone to increase MT density and detyrosination or in combination with parthenolide (10μM) to prevent the increase in detyrosination or HC-030031 (10μM) to block TRPA1. Voltage optical mapping was used to determine the nature of SIA. MT density was assessed by immunofluorescence in isolated LV myocytes. Results: Paclitaxel reduced the threshold for SIA (decreased V 50 versus control: 133±16 vs 173±11μL; n =8 , p =0.02 by paired t-test) and resulted in the conversion of some stretch-induced ectopy to sustained activity. These effects were prevented by reduced detyrosination with parthenolide (262±27 vs 256±26μL; n =8 , p =0.75). Paclitaxel also increased MT density versus control, however this was not prevented by parthenolide (63±3 and 60±3 vs 50±4%; n =15, p =0.04 by one-way ANOVA). The decreased V 50 and occurrence of sustained arrhythmias with paclitaxel was also prevented by TRPA1 block with HC-030031 (249±26 vs 237±27μL; n =8, p =0.60). Optical mapping revealed stretch-induced ectopic excitation originating from the LV freewall, which resulted in re-entrant activity with paclitaxel. Conclusions: An increase in MT detyrosination leads to a reduced threshold for SIA and the triggering of sustained arrhythmias in the whole heart in a TRPA1 dependent manner.

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