Calmodulin Interactions with Voltage-Gated Sodium Channels

Calmodulin (CaM) is a small protein that acts as a ubiquitous signal transducer and regulates neuronal plasticity, muscle contraction, and immune response. It interacts with ion channels and plays regulatory roles in cellular electrophysiology. CaM modulates the voltage-gated sodium channel gating process, alters sodium current density, and regulates sodium channel protein trafficking and expression. Many mutations in the CaM-binding IQ domain give rise to diseases including epilepsy, autism, and arrhythmias by interfering with CaM interaction with the channel. In the present review, we discuss CaM interactions with the voltage-gated sodium channel and modulators involved in CaM regulation, as well as summarize CaM-binding IQ domain mutations associated with human diseases in the voltage-gated sodium channel family.

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

Functional basis for calmodulation of the TRPV5 calcium channel

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+-selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic bilobal CaM regulation and binding stoichiometry. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+, and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without CaM N-lobe calcification. Moreover, we reveal a 1:2 stoichiometry of TRPV5:CaM binding by implementing single molecule photobleaching counting, a technique with great potential for studying TRP channel regulation. In conclusion, our study proposes a new model for CaM- dependent regulation – calmodulation – of the Ca2+-selective TRPV5 that involves apoCaM interaction and lobe-specific actions.

Complementary and alternative medicine - practice, attitudes, and knowledge among healthcare professionals in New Zealand: an integrative review

Background The prevalence of CAM use is increasing. This integrative review investigated New Zealand healthcare professionals’ practice of, attitudes toward, and knowledge about complementary and alternative medicine (CAM). Methods Literature search was conducted in four databases from inception to April 2020. Studies were included if they reported results from primary data collection on practice of, attitudes toward, or knowledge about CAM amongst New Zealand healthcare professionals. Results Eleven studies (two of ‘high-quality’, seven of ‘moderate-quality’, and two of ‘low-quality’) were identified with 2060 healthcare professionals including general practitioners (GPs), nurses, midwives, pharmacists, physiotherapists, and medical specialists. New Zealand healthcare professionals were generally positive regarding CAM use, but have concerns on the scientific evidence, regulation, safety, financial costs of CAM, and encourage an evidence-based CAM practice and stronger CAM regulation. Findings indicated that around 25% of GPs practise CAM, and 82.3% refer patients to CAM practitioners. When treating pregnant women, 48.4% of physiotherapists practise acupuncture, and 37.3% of midwives recommend CAM. GPs believe that acupuncture is the most helpful CAM modality, and most commonly practiced and referred patients to acupuncture. Up to 58% of GPs and Plunket nurses wanted to receive further education on CAM, and up to 66.7% GPs favour the idea CAM should be included in medical curriculums. Conclusions Nine of the 11 included studies were of moderate to high quality, thus enhancing the reliability of the review findings. In order to better manage CAM in New Zealand New Zealand clinical settings, there is a need to invest in CAM research and education, and enhance CAM regulation. This review is a first step in developing an evidence base to offer insights for further development of effective CAM policies regarding safety, efficacy, regulation and integration in New Zealand.

The S2–S3 Loop of Kv7.4 Channels Is Essential for Calmodulin Regulation of Channel Activation

Kv7.4 (KCNQ4) voltage-gated potassium channels control excitability in the inner ear and the central auditory pathway. Mutations in Kv7.4 channels result in inherited progressive deafness in humans. Calmodulin (CaM) is crucial for regulating Kv7 channels, but how CaM affects Kv7 activity has remained unclear. Here, based on electrophysiological recordings, we report that the third EF hand (EF3) of CaM controls the calcium-dependent regulation of Kv7.4 activation and that the S2–S3 loop of Kv7.4 is essential for the regulation mediated by CaM. Overexpression of the mutant CaM1234, which loses the calcium binding ability of all four EF hands, facilitates Kv7.4 activation by accelerating activation kinetics and shifting the voltage dependence of activation leftwards. The single mutant CaM3, which loses the calcium binding ability of the EF3, phenocopies facilitating effects of CaM1234 on Kv7.4 activation. Kv7.4 channels co-expressed with wild-type (WT) CaM show inhibited activation when intracellular calcium levels increase, while Kv7.4 channels co-expressed with CaM1234 or CaM3 are insensitive to calcium. Mutations C156A, C157A, C158V, R159, and R161A, which are located within the Kv7.4 S2–S3 loop, dramatically facilitate activation of Kv7.4 channels co-expressed with WT CaM but have no effect on activation of Kv7.4 channels co-expressed with CaM3, indicating that these five mutations decrease the inhibitory effect of Ca2+/CaM. The double mutation C156A/R159A decreases Ca2+/CaM binding and completely abolishes CaM-mediated calcium-dependent regulation of Kv7.4 activation. Taken together, our results provide mechanistic insights into CaM regulation of Kv7.4 activation and highlight the crucial role of the Kv7.4 S2–S3 loop in CaM regulation.

CaV channels reject signaling from a second CaM in eliciting Ca2+-dependent feedback regulation

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca2+ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaMs to the CaV nanodomain by tethering either WT or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary β2A subunit. We observed that a single CaM tethered to either the α or β2A subunit tunes Ca2+ regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit–tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca2+ feedback of CaV channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-dependent feedback of channel gating but may support alternate regulatory functions.

Transcript and metabolite changes during the early phase of abscisic acid-mediated induction of crassulacean acid metabolism in Talinum triangulare

Crassulacean acid metabolism (CAM) has evolved as a water-saving strategy, and its engineering into crops offers an opportunity to improve their water use efficiency. This requires a comprehensive understanding of the regulation of the CAM pathway. Here, we use the facultative CAM species Talinum triangulare as a model in which CAM can be induced rapidly by exogenous abscisic acid. RNA sequencing and metabolite measurements were employed to analyse the changes underlying CAM induction and identify potential CAM regulators. Non-negative matrix factorization followed by k-means clustering identified an early CAM-specific cluster and a late one, which was specific for the early light phase. Enrichment analysis revealed abscisic acid metabolism, WRKY-regulated transcription, sugar and nutrient transport, and protein degradation in these clusters. Activation of the CAM pathway was supported by up-regulation of phosphoenolpyruvate carboxylase, cytosolic and chloroplastic malic enzymes, and several transport proteins, as well as by increased end-of-night titratable acidity and malate accumulation. The transcription factors HSFA2, NF-YA9, and JMJ27 were identified as candidate regulators of CAM induction. With this study we promote the model species T. triangulare, in which CAM can be induced in a controlled way, enabling further deciphering of CAM regulation.

Allosteric regulators selectively prevent Ca2+-feedback of CaV and NaV channels

Calmodulin (CaM) serves as a pervasive regulatory subunit of CaV1, CaV2, and NaV1 channels, exploiting a functionally conserved carboxy-tail element to afford dynamic Ca2+-feedback of cellular excitability in neurons and cardiomyocytes. Yet this modularity counters functional adaptability, as global changes in ambient CaM indiscriminately alter its targets. Here, we demonstrate that two structurally unrelated proteins, SH3 and cysteine-rich domain (stac) and fibroblast growth factor homologous factors (fhf) selectively diminish Ca2+/CaM-regulation of CaV1 and NaV1 families, respectively. The two proteins operate on allosteric sites within upstream portions of respective channel carboxy-tails, distinct from the CaM-binding interface. Generalizing this mechanism, insertion of a short RxxK binding motif into CaV1.3 carboxy-tail confers synthetic switching of CaM regulation by Mona SH3 domain. Overall, our findings identify a general class of auxiliary proteins that modify Ca2+/CaM signaling to individual targets allowing spatial and temporal orchestration of feedback, and outline strategies for engineering Ca2+/CaM signaling to individual targets.

Calmodulin: A Multitasking Protein in Kv7.2 Potassium Channel Functions

The ubiquitous calcium transducer calmodulin (CaM) plays a pivotal role in many cellular processes, regulating a myriad of structurally different target proteins. Indeed, it is unquestionable that CaM is the most relevant transductor of calcium signals in eukaryotic cells. During the last two decades, different studies have demonstrated that CaM mediates the modulation of several ion channels. Among others, it has been indicated that Kv7.2 channels, one of the members of the voltage gated potassium channel family that plays a critical role in brain excitability, requires CaM binding to regulate the different mechanisms that govern its functions. The purpose of this review is to provide an overview of the most recent advances in structure–function studies on the role of CaM regulation of Kv7.2 and the other members of the Kv7 family.

Governing complementary and alternative medicine (CAM) in Brazil and Portugal: implications for CAM professionals and the public

Sociological research on the governance of complementary and alternative medicine (CAM) in Western societies has vastly increased in the last decades. Yet there has been a less marked expression of qualitative studies which put such governance into comparative perspective. Furthermore, research has shown that CAM regulation in Western countries has been very diverse, and so is probably best conceptualised on a spectrum containing several regulatory models. This chapter investigates CAM’s modes of governance in two historically, culturally and politically related countries, Brazil and Portugal. It analyses the extent to which CAM governance has changed over time in these two countries, the main modes of CAM governance in these same countries, and the implications of these modes of CAM governance for CAM professionals themselves and the public. It is concluded that Brazil and Portugal present some similar patterns in the way they govern CAM, but also contrasting differences, particularly in relation to the status of these therapies within the public and the private health care systems, and the implications of this status for CAM professionals themselves and the wider public.