Browsing by Subject "Calcium channel"

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    Complex Arrhythmia Syndrome in a Knock-In Mouse Model Carrier of the N98S Calm1 Mutation
    (American Heart Association, 2020) Tsai, Wen-Chin; Guo, Shuai; Olaopa, Michael A.; Field, Loren J.; Yang, Jin; Shen, Changyu; Chang, Ching-Pin; Chen, Peng-Sheng; Rubart, Michael; Medicine, School of Medicine
    Background: Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in CALM1 resulting in a p.N98S substitution in a patient with sinus bradycardia and stress-induced bidirectional ventricular ectopy. The objectives of the present study were to determine if mice carrying the N98S mutation knocked into Calm1 replicate the human arrhythmia phenotype and to examine arrhythmia mechanisms. Methods: Mouse lines heterozygous for the Calm1N98S allele (Calm1N98S/+) were generated using CRISPR/Cas9 technology. Adult mutant mice and their wildtype littermates (Calm1+/+) underwent electrocardiographic monitoring. Ventricular de- and repolarization was assessed in isolated hearts using optical voltage mapping. Action potentials and whole-cell currents and [Ca2+]i, as well, were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was used for in situ membrane voltage monitoring of ventricular conduction fibers. Results: Two biologically independent knock-in mouse lines heterozygous for the Calm1N98S allele were generated. Calm1N98S/+ mice of either sex and line exhibited sinus bradycardia, QTc interval prolongation, and catecholaminergic bidirectional ventricular tachycardia. Male mutant mice also showed QRS widening. Pharmacological blockade and activation of β-adrenergic receptors rescued and exacerbated, respectively, the long-QT phenotype of Calm1N98S/+ mice. Optical and electric assessment of membrane potential in isolated hearts and single left ventricular myocytes, respectively, revealed β-adrenergically induced delay of repolarization. β-Adrenergic stimulation increased peak density, slowed inactivation, and left-shifted the activation curve of ICa.L significantly more in Calm1N98S/+ versus Calm1+/+ ventricular myocytes, increasing late ICa.L in the former. Rapidly paced Calm1N98S/+ ventricular myocytes showed increased propensity to delayed afterdepolarization-induced triggered activity, whereas in situ His-Purkinje fibers exhibited increased susceptibility for pause-dependent early afterdepolarizations. Epicardial mapping of Calm1N98S/+ hearts showed that both reentry and focal mechanisms contribute to arrhythmogenesis. Conclusions: Heterozygosity for the Calm1N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QTc interval prolongation, and bidirectional ventricular tachycardia. β-Adrenergically induced ICa.L dysregulation contributes to the long-QT phenotype. Pause-dependent early afterdepolarizations and tachycardia-induced delayed afterdepolarizations originating in the His-Purkinje network and ventricular myocytes, respectively, constitute potential sources of arrhythmia in Calm1N98S/+ hearts.
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    In Vivo Identification and Manipulation of the Ca2+ Selectivity Filter in the Drosophila Transient Receptor Potential Channel
    (Society for Neuroscience, 2007-01-17) Liu, Che H.; Wang, Tao; Postma, Marten; Obukhov, Alexander G.; Montell, Craig; Hardie, Roger C.; Cellular and Integrative Physiology, School of Medicine
    Null mutations in the transient receptor potential (trp) gene eliminate the major, Ca2+-selective component of the light-sensitive conductance in Drosophila photoreceptors. Although it is the prototypical member of the TRP ion channel superfamily, conclusive evidence that TRP is a pore-forming channel subunit in vivo is lacking. We show here that mutating a specific acidic residue (Asp621) in the putative pore virtually eliminated Ca2+ permeation in vivo and altered other biophysical properties of the native TRP conductance. The results identify Asp621 as a critical residue of the TRP Ca2+ selectivity filter, provide the first rigorous demonstration that a TRP protein is a pore-forming subunit in any native system, and point to the likely location of the pore in mammalian canonical TRP channels. The specific elimination of Ca2+ permeation in TRP also provided a unique opportunity to address the roles of Ca2+ influx in vivo. We found that Asp621 mutations profoundly affected several key aspects of the light response and caused light-dependent retinal degeneration.
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    Small-molecule CaVα1⋅CaVβ antagonist suppresses neuronal voltage-gated calcium-channel trafficking
    (National Academy of Sciences, 2018-11-06) Chen, Xingjuan; Liu, Degang; Zhou, Donghui; Si, Yubing; Xu, David; Stamatkin, Christopher W.; Ghozayel, Mona K.; Ripsch, Matthew S.; Obukhov, Alexander G.; White, Fletcher A.; Meroueh, Samy O.; Cellular and Integrative Physiology, School of Medicine
    Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1 subunit (CaVα1) that is engaged in protein-protein interactions with auxiliary α2/δ and β subunits. The high-affinity CaV2.2α1⋅CaVβ3 protein-protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1⋅CaVβ3 protein-protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1 Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVβ3 Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein-protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.