Competitive Tuning Among Ca2+/Calmodulin-Dependent Proteins: Analysis of in silico Model Robustness and Parameter Variability.
Ontology highlight
ABSTRACT:
SUBMITTER: Pharris MC
PROVIDER: S-EPMC6519966 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2+) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca2+/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca2+ signaling pathways in neurons. It was shown experimentally that Ca2+/CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca2+-free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng13-49), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng13-49 structures by matching the changes in the chemical shifts of CaM upon Ng13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca2+ for CaM in the presence of Ng13-49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca2+ binding loops particularly at C-domain of CaM, enabling Ca2+ release. In contrast, CaMKII peptide increases Ca2+ affinity for the C-domain of CaM by stabilizing the two Ca2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca2+ binding affinities.
| S-EPMC5374985 | biostudies-literature
Project description:Penalized regression methods that perform simultaneous model selection and estimation are ubiquitous in statistical modeling. The use of such methods is often unavoidable as manual inspection of all possible models quickly becomes intractable when there are more than a handful of predictors. However, automated methods usually fail to incorporate domain-knowledge, exploratory analyses, or other factors that might guide a more interactive model-building approach. A hybrid approach is to use penalized regression to identify a set of candidate models and then to use interactive model-building to examine this candidate set more closely. To identify a set of candidate models, we derive point and interval estimators of the probability that each model along a solution path will minimize a given model selection criterion, for example, Akaike information criterion, Bayesian information criterion (AIC, BIC), etc., conditional on the observed solution path. Then models with a high probability of selection are considered for further examination. Thus, the proposed methodology attempts to strike a balance between algorithmic modeling approaches that are computationally efficient but fail to incorporate expert knowledge, and interactive modeling approaches that are labor intensive but informed by experience, intuition, and domain knowledge. Supplementary materials for this article are available online.
| S-EPMC6750234 | biostudies-literature
Project description:The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca2+-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca2+-dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca2+ concentrations above 0.2 µM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca2+ saturated under resting Ca2+ conditions. Conversely, the N-domain senses Ca2+ exactly in the transition from resting to activating Ca2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca2+ during Ca2+ oscillations, while the CaM N-domain functions as a dynamic Ca2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.
| S-EPMC6340113 | biostudies-literature
Project description:Transient receptor potential (TRP) polycystin-3 (TRPP3) is a non-selective cation channel activated by Ca2+ and protons and is involved in regulating ciliary Ca2+ concentration, hedgehog signaling and sour tasting. The TRPP3 channel function and regulation are still not well understood. Here we investigated regulation of TRPP3 by calmodulin (CaM) by means of electrophysiology and Xenopus oocytes as an expression model. We found that TRPP3 channel function is enhanced by calmidazolium, a CaM antagonist, and inhibited by CaM through binding of the CaM N-lobe to a TRPP3 C-terminal domain not overlapped with the EF-hand. We further revealed that the TRPP3/CaM interaction promotes phosphorylation of TRPP3 at threonine 591 by Ca2+/CaM-dependent protein kinase II, which mediates the inhibition of TRPP3 by CaM.
| S-EPMC10134200 | biostudies-literature
Project description:Ca(2+) channels and calmodulin (CaM) are two prominent signalling hubs that synergistically affect functions as diverse as cardiac excitability, synaptic plasticity and gene transcription. It is therefore fitting that these hubs are in some sense coordinated, as the opening of Ca(V)1-2 Ca(2+) channels are regulated by a single CaM constitutively complexed with channels. The Ca(2+)-free form of CaM (apoCaM) is already pre-associated with the isoleucine-glutamine (IQ) domain on the channel carboxy terminus, and subsequent Ca(2+) binding to this 'resident' CaM drives conformational changes that then trigger regulation of channel opening. Another potential avenue for channel-CaM coordination could arise from the absence of Ca(2+) regulation in channels lacking a pre-associated CaM. Natural fluctuations in CaM concentrations might then influence the fraction of regulable channels and, thereby, the overall strength of Ca(2+) feedback. However, the prevailing view has been that the ultrastrong affinity of channels for apoCaM ensures their saturation with CaM, yielding a significant form of concentration independence between Ca(2+) channels and CaM. Here we show that significant exceptions to this autonomy exist, by combining electrophysiology (to characterize channel regulation) with optical fluorescence resonance energy transfer (FRET) sensor determination of free-apoCaM concentration in live cells. This approach translates quantitative CaM biochemistry from the traditional test-tube context into the realm of functioning holochannels within intact cells. From this perspective, we find that long splice forms of Ca(V)1.3 and Ca(V)1.4 channels include a distal carboxy tail that resembles an enzyme competitive inhibitor that retunes channel affinity for apoCaM such that natural CaM variations affect the strength of Ca(2+) feedback modulation. Given the ubiquity of these channels, the connection between ambient CaM levels and Ca(2+) entry through channels is broadly significant for Ca(2+) homeostasis. Strategies such as ours promise key advances for the in situ analysis of signalling molecules resistant to in vitro reconstitution, such as Ca(2+) channels.
| S-EPMC3553577 | biostudies-literature
Project description:N-methyl-d-aspartate (NMDA) receptors are glutamate- and glycine-gated channels that flux Na+ and Ca2+ into postsynaptic neurons during synaptic transmission. The resulting intracellular Ca2+ transient is essential to physiological and pathological processes related to synaptic development, plasticity, and apoptosis. It also engages calmodulin (CaM) to reduce subsequent NMDA receptor activity in a process known as Ca2+-dependent inactivation (CDI). Here, we used whole-cell electrophysiology to measure CDI and computational modeling to dissect the sequence of events that underlies it. With these approaches, we estimate that CaM senses NMDA receptor Ca2+ influx at ?9 nm from the channel pore. Further, when we controlled the frequency of Ca2+ influx through individual channels, we found that a kinetic model where apoCaM associates with channels before their activation best predicts the measured CDI. These results provide, to our knowledge, novel functional evidence for CaM preassociation to NMDA receptors in living cells. This particular mechanism for autoinhibitory feedback reveals strategies and challenges for Ca2+ regulation in neurons during physiological synaptic activity and disease.
| S-EPMC5700250 | biostudies-literature
Project description:KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+-binding protein calmodulin (CaM), thereby providing an intersection between KSR1-mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca2+/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca2+/CaM to a globular conformation of KSR1-CA3 via electrostatic interactions or (2) binding of collapsed Ca2+/CaM to α-helical KSR1-CA3 via hydrophobic interactions. Experimentally, site-directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca2+/CaM binds to the α-helical conformation of KSR1-CA3. Importantly, replacing KSR1-Phe355 with Asp reduces Ca2+/CaM binding by 76%. The KSR1-F355D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca2+/CaM binding promotes KSR1-mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca2+/CaM, identifies a KSR1 single-point mutant as a bioreagent to selectively study the crosstalk between Ca2+ and KSR1-mediated signaling.
| S-EPMC11002989 | biostudies-literature
Project description:The antidepressant-sensitive norepinephrine (NE) transporter (NET) inactivates NE released during central and peripheral neuronal activity by transport into presynaptic cells. Altered NE clearance due to dysfunction of NET has been associated with the development of mental illness and cardiovascular diseases. NET activity in vivo is influenced by stress, neuronal activity, hormones and drugs. We investigated the mechanisms of Ca2+ regulation of NET and found that Ca2+ influenced both Vmax and Km for NE transport into cortical synaptosomes. Changes in extracellular Ca2+ triggered rapid and bidirectional surface trafficking of NET expressed in cultured cells. Deletion of residues 28-47 in the NET NH2-terminus abolished the Ca2+ effect on surface trafficking. Mutagenesis studies identified Thr30 in this region as the essential residue for both Ca2+- dependent phosphorylation and trafficking of NET. Depolarization of excitable cells increased surface NET in a Thr30 dependent manner. A proteomic analysis, RNA interference, and pharmacological inhibition supported roles of CaMKI and CaMKII in Ca2+-modulated NE transport and NET trafficking. Depolarization of primary noradrenergic neurons in culture with elevated K+ increased NET surface expression in a process that required external Ca2+ and depended on CaMK activity. Hippocampal NE clearance in vivo was also stimulated by depolarization, and inhibitors of CaMK signaling prevented this stimulation. In summary, Ca2+ signaling influenced surface trafficking of NET through a CaMK-dependent mechanism requiring Thr30.
| S-EPMC5481502 | biostudies-literature
Project description:Background Oxidative stress-mediated Ca2+/calmodulin-dependent protein kinase II (Ca MKII) phosphorylation of cardiac ion channels has emerged as a critical contributor to arrhythmogenesis in cardiac pathology. However, the link between mitochondrial-derived reactive oxygen species (md ROS ) and increased Ca MKII activity in the context of cardiac arrhythmias has not been fully elucidated and is difficult to establish experimentally. Methods and Results We hypothesize that pathological md ROS can cause erratic action potentials through the oxidation-dependent Ca MKII activation pathway. We further propose that Ca MKII -dependent phosphorylation of sarcolemmal slow Na+ channels alone is sufficient to elicit early afterdepolarizations. To test the hypotheses, we expanded our well-established guinea pig cardiomyocyte excitation- contraction coupling, mitochondrial energetics, and ROS - induced- ROS - release model by incorporating oxidative Ca MKII activation and Ca MKII -dependent Na+ channel phosphorylation in silico. Simulations show that md ROS mediated-Ca MKII activation elicits early afterdepolarizations by augmenting the late Na+ currents, which can be suppressed by blocking L-type Ca2+ channels or Na+/Ca2+ exchangers. Interestingly, we found that oxidative Ca MKII activation-induced early afterdepolarizations are sustained even after md ROS has returned to its physiological levels. Moreover, mitochondrial-targeting antioxidant treatment can suppress the early afterdepolarizations, but only if given in an appropriate time window. Incorporating concurrent md ROS -induced ryanodine receptors activation further exacerbates the proarrhythmogenic effect of oxidative Ca MKII activation. Conclusions We conclude that oxidative Ca MKII activation-dependent Na channel phosphorylation is a critical pathway in mitochondria-mediated cardiac arrhythmogenesis.
| S-EPMC6201444 | biostudies-literature
Project description:TRPA1 is a Ca2+-permeable ion channel involved in many sensory disorders such as pain, itch and neuropathy. Notably, the function of TRPA1 depends on Ca2+, with low Ca2+ potentiating and high Ca2+ inactivating TRPA1. However, it remains unknown how Ca2+ exerts such contrasting effects. Here, we show that Ca2+ regulates TRPA1 through calmodulin, which binds to TRPA1 in a Ca2+-dependent manner. Calmodulin binding enhanced TRPA1 sensitivity and Ca2+-evoked potentiation of TRPA1 at low Ca2+, but inhibited TRPA1 sensitivity and promoted TRPA1 desensitization at high Ca2+. Ca2+-dependent potentiation and inactivation of TRPA1 were selectively prevented by disrupting the interaction of the carboxy-lobe of calmodulin with a calmodulin-binding domain in the C-terminus of TRPA1. Calmodulin is thus a critical Ca2+ sensor enabling TRPA1 to respond to diverse Ca2+ signals distinctly.
| S-EPMC5362816 | biostudies-literature