Elementary mechanisms of calmodulin regulation of Na1.5 producing divergent arrhythmogenic phenotypes.

In cardiomyocytes, Na1.5 channels mediate initiation and fast propagation of action potentials. The Ca-binding protein calmodulin (CaM) serves as a de facto subunit of Na1.

The molecular basis of the inhibition of Ca1 calcium-dependent inactivation by the distal carboxy tail.

Ca/calmodulin-dependent inactivation (CDI) of Ca channels is a critical regulatory process that tunes the kinetics of Ca entry for different cell types and physiologic responses. CDI is mediated by calmodulin (CaM), which is bound to the IQ domain of the Ca carboxy tail. This modulatory process is tailored by alternative splicing such that select splice variants of Ca1.

Evaluation of referrals for short stature: A retrospective chart review.

Background: Referrals to paediatric endocrine clinics for short stature are common. Height velocity (HV) is an essential component of the evaluation of short stature as growth deceleration often reflects an underlying paediatric endocrine diagnosis (PED). Access to previous measurements facilitates prompt calculation of HV.

A bilobal model of Ca-dependent inactivation to probe the physiology of L-type Ca channels.

L-type calcium channels (LTCCs) are critical elements of normal cardiac function, playing a major role in orchestrating cardiac electrical activity and initiating downstream signaling processes. LTCCs thus use feedback mechanisms to precisely control calcium (Ca) entry into cells. Of these, Ca-dependent inactivation (CDI) is significant because it shapes cardiac action potential duration and is essential for normal cardiac rhythm.

Allosteric regulators selectively prevent Ca-feedback of Ca and Na channels.

Calmodulin (CaM) serves as a pervasive regulatory subunit of Ca1, Ca2, and Na1 channels, exploiting a functionally conserved carboxy-tail element to afford dynamic Ca-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 Ca/CaM-regulation of Ca1 and Na1 families, respectively.

Food allergy and anaphylaxis.

Anaphylaxis is a severe and potentially life-threatening allergic reaction. There are numerous potential causes, with food allergy being the leading cause in children and the focus of this review. Most reactions involve an IgE-mediated mechanism, although non-IgE-mediated and nonimmunologic reactions can occur.

Duplex signaling by CaM and Stac3 enhances Ca1.1 function and provides insights into congenital myopathy.

Ca1.1 is essential for skeletal muscle excitation-contraction coupling. Its functional expression is tuned by numerous regulatory proteins, yet underlying modulatory mechanisms remain ambiguous as Ca1.

Electromechanics and Volume Dynamics in Nonexcitable Tissue Cells.

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell's lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment.

Conservation of cardiac L-type Ca channels and their regulation in Drosophila: A novel genetically-pliable channelopathic model.

Dysregulation of L-type Ca channels (LTCCs) underlies numerous cardiac pathologies. Understanding their modulation with high fidelity relies on investigating LTCCs in their native environment with intact interacting proteins. Such studies benefit from genetic manipulation of endogenous channels in cardiomyocytes, which often proves cumbersome in mammalian models.

Bilobal architecture is a requirement for calmodulin signaling to Ca1.3 channels.

Calmodulin (CaM) regulation of voltage-gated calcium (Ca) channels is a powerful Ca feedback mechanism that adjusts Ca influx, affording rich mechanistic insights into Ca decoding. CaM possesses a dual-lobed architecture, a salient feature of the myriad Ca-sensing proteins, where two homologous lobes that recognize similar targets hint at redundant signaling mechanisms. Here, by tethering CaM lobes, we demonstrate that bilobal architecture is obligatory for signaling to Ca channels.

Multiscale mapping of frequency sweep rate in mouse auditory cortex.

Functional organization is a key feature of the neocortex that often guides studies of sensory processing, development, and plasticity. Tonotopy, which arises from the transduction properties of the cochlea, is the most widely studied organizational feature in auditory cortex; however, in order to process complex sounds, cortical regions are likely specialized for higher order features. Here, motivated by the prevalence of frequency modulations in mouse ultrasonic vocalizations and aided by the use of a multiscale imaging approach, we uncover a functional organization across the extent of auditory cortex for the rate of frequency modulated (FM) sweeps.

Detecting stoichiometry of macromolecular complexes in live cells using FRET.

The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex.

Quantifying macromolecular interactions in living cells using FRET two-hybrid assays.

Förster resonance energy transfer (FRET) is a versatile method for analyzing protein-protein interactions within living cells. This protocol describes a nondestructive live-cell FRET assay for robust quantification of relative binding affinities for protein-protein interactions. Unlike other approaches, our method correlates the measured FRET efficiencies to relative concentration of interacting proteins to determine binding isotherms while including collisional FRET corrections.

A Precision Medicine Approach to the Rescue of Function on Malignant Calmodulinopathic Long-QT Syndrome.

Rationale: Calmodulinopathies comprise a new category of potentially life-threatening genetic arrhythmia syndromes capable of producing severe long-QT syndrome (LQTS) with mutations involving CALM1, CALM2, or CALM3. The underlying basis of this form of LQTS is a disruption of Ca/calmodulin (CaM)-dependent inactivation of L-type Ca channels.

Objective: To gain insight into the mechanistic underpinnings of calmodulinopathies and devise new therapeutic strategies for the treatment of this form of LQTS.

Protein kinase A modulation of CaV1.4 calcium channels.

The regulation of L-type Ca(2+) channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca(2+) channels, relatively little is known about the closely related CaV1.

An autism-associated mutation in CaV1.3 channels has opposing effects on voltage- and Ca(2+)-dependent regulation.

CaV1.3 channels are a major class of L-type Ca(2+) channels which contribute to the rhythmicity of the heart and brain. In the brain, these channels are vital for excitation-transcription coupling, synaptic plasticity, and neuronal firing.

Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET.

Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer.

Arrhythmogenesis in Timothy Syndrome is associated with defects in Ca(2+)-dependent inactivation.

Timothy Syndrome (TS) is a multisystem disorder, prominently featuring cardiac action potential prolongation with paroxysms of life-threatening arrhythmias. The underlying defect is a single de novo missense mutation in CaV1.2 channels, either G406R or G402S.

Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes.

Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells.

Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels.

Voltage-gated Na and Ca(2+) channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge.

Large Ca²⁺-dependent facilitation of Ca(V)2.1 channels revealed by Ca²⁺ photo-uncaging.

Key Points: CaV 2.1 channels constitute a dominant Ca(2+) entry pathway into brain neurons, triggering downstream Ca(2+) -dependent processes such as neurotransmitter release. CaV 2.

Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation.

The Ca(2+)-free form of calmodulin (apoCaM) often appears inert, modulating target molecules only upon conversion to its Ca(2+)-bound form. This schema has appeared to govern voltage-gated Ca(2+) channels, where apoCaM has been considered a dormant Ca(2+) sensor, associated with channels but awaiting the binding of Ca(2+) ions before inhibiting channel opening to provide vital feedback inhibition. Using single-molecule measurements of channels and chemical dimerization to elevate apoCaM, we find that apoCaM binding on its own markedly upregulates opening, rivaling the strongest forms of modulation.

Optical mapping of optogenetically shaped cardiac action potentials.

Light-mediated silencing and stimulation of cardiac excitability, an important complement to electrical stimulation, promises important discoveries and therapies. To date, cardiac optogenetics has been studied with patch-clamp, multielectrode arrays, video microscopy, and an all-optical system measuring calcium transients. The future lies in achieving simultaneous optical acquisition of excitability signals and optogenetic control, both with high spatio-temporal resolution.

Calcineurin determines toxic versus beneficial responses to α-synuclein.

Calcineurin (CN) is a highly conserved Ca(2+)-calmodulin (CaM)-dependent phosphatase that senses Ca(2+) concentrations and transduces that information into cellular responses. Ca(2+) homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca(2+), thereby activating a CaM-CN cascade that engages substrates that result in toxicity.

Multiscale optical Ca2+ imaging of tonal organization in mouse auditory cortex.

Spatial patterns of functional organization, resolved by microelectrode mapping, comprise a core principle of sensory cortices. In auditory cortex, however, recent two-photon Ca2+ imaging challenges this precept, as the traditional tonotopic arrangement appears weakly organized at the level of individual neurons. To resolve this fundamental ambiguity about the organization of auditory cortex, we developed multiscale optical Ca2+ imaging of unanesthetized GCaMP transgenic mice.