A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels.

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on ß-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavß subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVß, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVß, is sufficient for sympathetic upregulation of Ca2+ currents.

Asymmetric contribution of a selectivity filter gate in triggering inactivation of CaV1.3 channels.

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultrahighly conserved tryptophan residues in the interdomain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudotetrameric CaV pore domain for feedback inhibition.

Asymmetric Contribution of a Selectivity Filter Gate in Triggering Inactivation of CaV1.3 Channels.

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultra-highly conserved tryptophan residues in the inter-domain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudo-tetrameric CaV pore domain for feedback inhibition.

A Genetically Encoded Actuator Selectively Boosts L-type Calcium Channels in Diverse Physiological Settings.

L-type Ca 2+ channels (Ca V 1.2/1.3) convey influx of calcium ions (Ca 2+ ) that orchestrate a bevy of biological responses including muscle contraction and gene transcription. Deficits in Ca V 1 function play a vital role in cardiac and neurodevelopmental disorders. Yet conventional pharmacological approaches to upregulate Ca V 1 are limited, as excessive Ca 2+ influx leads to cytotoxicity. Here, we develop a genetically encoded enhancer of Ca V 1.2/1.3 channels (GeeC) to manipulate Ca 2+ entry in distinct physiological settings. Specifically, we functionalized a nanobody that targets the Ca V macromolecular complex by attaching a minimal effector domain from a Ca V enhancer-leucine rich repeat containing protein 10 (Lrrc10). In cardiomyocytes, GeeC evoked a 3-fold increase in L-type current amplitude. In neurons, GeeC augmented excitation-transcription (E-T) coupling. In all, GeeC represents a powerful strategy to boost Ca V 1.2/1.3 function in distinct physiological settings and, in so doing, lays the groundwork to illuminate new insights on neuronal and cardiac physiology and disease.

Sympathetic Nervous System Regulation of Cardiac Calcium Channels.

Calcium influx through voltage-gated calcium channels, Cav1.2, in cardiomyocytes initiates excitation-contraction coupling in the heart. The force and rate of cardiac contraction are modulated by the sympathetic nervous system, mediated substantially by changes in intracellular calcium. Norepinephrine released from sympathetic neurons innervating the heart and epinephrine secreted by the adrenal chromaffin cells bind to ß-adrenergic receptors on the sarcolemma of cardiomyocytes initiating a signaling cascade that generates cAMP and activates protein kinase A, the targets of which control calcium influx. For decades, the mechanisms by which PKA regulated calcium channels in the heart were not known. Recently, these mechanisms have been elucidated. In this chapter, we will review the history of the field and the studies that led to the identification of the evolutionarily conserved process.

Adrenergic CaV1.2 Activation via Rad Phosphorylation Converges at α1C I-II Loop.

RATIONALE: Changing activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic. OBJECTIVE: The primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during ß-adrenergic stimulation, and in heart failure. METHODS AND RESULTS: We generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and ß-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVß to α1C I-II loop and eliminated ß-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to ß-adrenergic agonists when reconstituted heterologously with ß2B and Rad or transgenically expressed in cardiomyocytes. CONCLUSIONS: Ca2+ channel activity is dynamically modulated under basal conditions, during ß-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVß binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the ß-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by ß-adrenergic agonists/PKA also requires this rigid linker and ß-binding to α1C.

Cutting out the fat: Site-specific deacylation of an ion channel.

S-Acylation, a reversible post-translational lipid modification of proteins, controls the properties and function of various proteins, including ion channels. Large conductance Ca2+-activated potassium (BK) channels are S-acylated at two sites that impart distinct functional effects. Whereas the enzymes that attach lipid groups are known, the enzymes mediating lipid removal (i.e. deacylation) are largely unknown. Here, McClafferty et al. identify two enzymes, ABHD17a and ABHD17c, that excise BK channel lipid groups with remarkable precision. These findings lend insights into mechanisms that orchestrate the (de)acylation that fine-tunes ion channel function in physiology and disease.

Anticoagulation Therapy by Age and Embolic Risk for Nonvalvular Atrial Fibrillation in Mexico, an Upper-Middle-Income Country: The CARMEN-AF Registry.

Background: Documenting the patterns of oral anticoagulation therapy (OAT) is essential to prevent thromboembolic complications of nonvalvular atrial fibrillation (NVAF). Objective: To report the patterns of OAT according to age and thromboembolic risk in patients included in CARMEN-AF, a nationwide registry of NVAF in Mexico, an upper middle-income country. Material and methods: There were 1,423 consecutive patients ≥18 years old and with at least one thromboembolic risk factor enrolled in the CARMEN-AF Registry at their regular clinical visit during a three-year period. They were analyzed according to 1) age, 2) AF type, and 3) CHA2DS2-VASc score. Results: Overall, 16.4% of patients did not receive antithrombotic treatment, 19.4% received antiplatelet drugs (APD), 29.2% vitamin K antagonists (VKA), and 34.6% direct oral anticoagulants (DOAC). With increasing age, the proportion of subjects treated with VKA decreased significantly from 36.2% in subjects <65 years to 22.5% in those ≥75 years old (P <0.0001). Concomitantly, an increase in both APD and no antithrombotic treatment was observed with increasing age. DOAC were prescribed equally among all age groups (34.2% in <65, 36.0% in 65-74, and 33.9% in ≥75). According to the type of AF, VKA use was more common in patients with permanent AF (32.7%). A lower use of DOAC was observed in high thromboembolic risk subjects (33.6% in CHA2DS2-VASc ≥2) compared with the moderate risk group (41% in CHA2DS2-VASc = 1). Conclusions: VKA use for NVAF in Mexico decreased in relation to increasing age. The proportion of DOAC therapy was the same in all age groups. Nevertheless, elderly patients with high thromboembolic risk received a suboptimal thromboprophylaxis. These data could help to improve gaps in the implementation of global guidelines. Clinical trial registration: http://www.clinicaltrials.gov. Unique identifier: NCT02334852. Highlights: CARMEN-AF is a nationwide multi-centric registry seeking to bridge the data gap on anticoagulation therapy for NVAF in Mexico.Elderly patients are more prone to receive suboptimal OAT for NVAF.DOAC were less frequently used in high thromboembolic risk patients (CHA2DS2-VASc ≥2).