Stewart, B. D. | Search bioRxiv

bioRxiv2018

Cation binding to SERCA

Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca2+) binds SERCA with micro-molar affinity, magnesium (Mg2+) and potassium (K+) also compete with calcium (Ca2+) binding. However, the molecular bases for these competing ions influence on SERCA function and the selectivity of the pump for Ca2+ are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca2+ binding sites: 1) triplicate molecular dynamics (MD) simulations of Mg2+, Ca2+and K+-bound SERCA. 2) mean spherical approximation (MSA) theory to determine the affinity and selectivity of cation binding to the MD-resolved structures and 3) state models of SERCA turnover informed from MSA-derived affinity data. Our key findings are that a) coordination at sites I and II are optimized for Ca2+and to a lesser extent for Mg2+ and K+, as determined by MD-derived cation-amino acid oxygen and bound water configurations, b) the impaired coordination and high desolvation cost for Mg2+ precludes favorable Mg2+ binding relative to Ca2+, while K+ has limited capacity to bind site I, c) Mg2+ most likely acts as inhibitor and K+ as intermediate in SERCAs reaction cycle, based on a best-fit state model of SERCA turnover. These findings provide a quantitative basis for SERCA function that leverages molecular-scale thermodynamic data and rationalize enzyme activity across broad ranges of K+, Ca2+ and Mg2+ concentrations.

biochemistry↗

bioRxiv2018

Simulation of P2X-mediated calcium signaling in microglia

Microglia function is orchestrated through highly-coupled signaling pathways that depend on calcium (Ca2+). In response to extracellular adenosine triphosphate (ATP), transient increases in intracellular Ca2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis and potentially their release. While steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic puriner-gic receptors, P2X4 and P2X7, with the graded production of a gene product, namely the tumor necrosis factor (TNF) cytokine. In addition to Ca2+ handling mechanisms common to eukaryotic cells, our model includes microglia-specific processes including ATP-dependent P2X4 and P2X7 activation, activation of NFAT transcription factors, and TNF production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNF production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNF production. Our key findings are that TNF production via P2X4 is maximized at low ATP when subject to high frequency ATP stimulation, whereas P2X7 contributes most significantly at millimolar ATPranges. Given that Ca2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.

systems biology↗