Regulation of cardiac physiology is well known to occur through the action of kinases that reversibly phosphorylate ion channels, calcium handling machinery, and signaling effectors. However, it is becoming increasingly apparent that palmitoylation or S-acylation, the post-translational modification of cysteines with saturated fatty acids, plays instrumental roles in regulating the localization, activity, stability, sorting, and function of numerous proteins, including proteins known to have essential functions in cardiomyocytes. However, the impact of this modification on cardiac physiology requires further investigation. S-acylation is catalyzed by the zDHHC family of S-acyl transferases that localize to intracellular organelle membranes or the sarcolemma. Recent work has begun to uncover functions of S-acylation in the heart, particularly in the regulation of cardiac electrophysiology, including modification of the sodiumcalcium exchanger, phospholemman and the cardiac sodium pump, as well as the voltage-gated sodium channel. Elucidating the regulatory functions of zDHHC enzymes in cardiomyocytes and determination of how S-acylation is altered in the diseased heart will shed light on how these modifications participate in cardiac pathogenesis and potentially identify novel targets for the treatment of cardiovascular disease. Indeed, proteins with critical signaling roles in the heart are also S-acylated, including receptors and G-proteins, yet the dynamics and functions of these modifications in myocardial physiology have not been interrogated. Here, we will review what is known about zDHHC enzymes and substrate S-acylation in myocardial physiology and highlight future areas of investigation that will uncover novel functions of S-acylation in cardiac homeostasis and pathophysiology.

Synaptotagmin‐7 (Syt‐7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin‐1 (Syt‐1). Despite a broad appreciation for the importance of Syt‐7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations—mouse chromaffin cells lacking endogenous Syt‐7 (KO cells) and a reconstituted system employing cell‐derived granules expressing either Syt‐7 or Syt‐1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt‐7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt‐7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt‐1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt‐7, granules expressing only Syt‐7 or Syt‐1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt‐7 confers substantially greater calcium sensitivity to granule fusion than Syt‐1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt‐7 plays a central role in regulating secretory output from adrenal chromaffin cells.

Protein palmitoylation is a critical posttranslational modification that regulates protein trafficking, localization, stability, sorting and function. In mammals, addition of this lipid modification onto proteins is mediated by a family of 23 palmitoyl acyl transferases (PATs). PATs often palmitoylate substrates in a promiscuous manner, precluding our understanding of how these enzymes achieve specificity for their substrates. Despite generous efforts to identify consensus motifs defining PAT-substrate specificity, it remains to be determined whether additional factors beyond interaction motifs, such as local palmitoylation, participate in PAT-substrate selection. In this review, we emphasize the role of local palmitoylation, in which substrates are palmitoylated and trapped in the same subcellular compartments as their PATs, as a mechanism of enzymesubstrate specificity. We focus here on non-Golgi-localized PATs, as physical proximity to their substrates enables them to engage in local palmitoylation, compared to Golgi PATs, which often direct trafficking of their substrates elsewhere. PAT subcellular localization may be an under-recognized, yet important determinant of PAT-substrate specificity that may work in conjunction or completely independently of interaction motifs. We also discuss some current hypotheses about protein motifs that contribute to localization of non-Golgi-localized PATs, important for the downstream targeting of their substrates.

Voltage-gated sodium channel (VGSC) β1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC α subunits and participate in cell–cell and cell–matrix adhesion, all with important implications for intracellular signal transduction, cell migration, and differentiation. Human loss-of-function variants in SCN1B, the gene encoding the VGSC β1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that β1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that β1 subunits undergo regulated intramembrane proteolysis (RIP) via the activity of β-secretase 1 (BACE1) and γ-secretase, resulting in the generation of a soluble intracellular domain, β1-ICD, which modulates transcription. Here, we report that β1 subunits are phosphorylated by FYN kinase. Moreover, we show that β1 subunits are S-palmitoylated. Substitution of a single residue in β1, Cys-162, to alanine prevented palmitoylation, reduced the level of β1 polypeptides at the plasma membrane, and reduced the extent of β1 RIP, suggesting that the plasma membrane is the site of β1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor Dyngo-4a restored plasma membrane association of β1-p.C162A to WT levels. Despite these observations, palmitoylation-null β1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration of S-palmitoylation of a VGSC β subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.

Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A, which encodes the sodium channel NaV1.2, and ANK2, which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how NaV1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding NaV1.2 to the dendritic membrane of mouse neocortical neurons, and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a+/- conditions. Thus, these results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can be etiological to neurodevelopmental disorder pathophysiology.