Fluxes of calcium (Ca2+) across cell membranes enable fast cellular responses. Calmodulin (CaM) senses local changes in Ca2+ concentration and relays the information to numerous interaction partners. The critical role of accurate Ca2+ signaling on cellular function is underscored by the fact that there are three independent CaM genes (CALM1-3) in the human genome. All three genes are functional and encode the exact same CaM protein. Moreover, CaM has a completely conserved amino acid sequence across all vertebrates. Given this degree of conservation, it was long thought that mutations in CaM were incompatible with life. It was therefore a big surprise when the first CaM mutations in humans were identified six years ago. Today, more than a dozen human CaM missense mutations have been described, all found in patients with severe cardiac arrhythmias. Biochemical studies have demonstrated differential effects on Ca2+ binding affinities for these CaM variants. Moreover, CaM regulation of central cardiac ion channels is impaired, including the voltage-gated Ca2+ channel, CaV1.2, and the sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor isoform 2, RyR2. Currently, no non-cardiac phenotypes have been described for CaM variant carriers. However, sequencing of large human cohorts reveals a cumulative frequency of additional rare CaM mutations that raise the possibility of CaM variants not exclusively causing severe cardiac arrhythmias. Here, we provide an overview of the identified CaM variants and their known consequences for target regulation and cardiac disease phenotype. We discuss experimental data, patient genotypes and phenotypes as well as which questions remain open to understand this complexity.

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in theCALMgenes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (CaV1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the CaV1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca2+by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca2+-free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca2+-dependent inactivation of the voltage-gated calcium channel.

Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general.

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.

Mutations in the genes encoding the highly conserved Ca2+-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca2+-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia–causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca2+ coordination and has only a minor impact on binding affinity toward Ca2+ and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca2+ channel, Cav1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca2+-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84–2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.