Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes

Abstract

Mammalian voltage-activated L-type Ca 2+ channels, such as Ca(v)1.2, control transmembrane Ca 2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current–voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca 2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca 2+ channels in excitable cells.

S-acylation of Ca2+ transport proteins: molecular basis and functional consequences

“…They control excitation-contraction coupling in muscle, neurotransmitter release in neurons, and insulin secretion by pancreatic beta cells [ 16 ]. The pore-forming α1C subunit of Ca v 1.2, a L-type voltage-dependent Ca 2+ channel encoded by the CACNA1C gene, was recently reported to be S-acylated in ventricular myocytes using resin-assisted capture of acylated proteins (Acyl-RAC) [ 17 ]. Acyl-RAC, along with acyl-biotin exchange and acyl-PEG exchange, rely on the breaking of disulfuric bonds, blocking of the free cysteines and cleaving of the S-acylated cysteine [ 18 ].…”

S‐acylation: an orchestrator of the life cycle and function of membrane proteins

“…S-acylation is dynamic, but the duration of the cycles varies depending on the protein and the physiological state of the cell, from minutes to days-the latter one being quasi-equivalent to irreversible on the cellular time scale [58,59]. Removal of the acyl chain is carried out by ); conformational changes (e.g., voltage-gated channels [14][15][16]); multimerization (e.g., STING [34,35], Gasdermin D pores [36,37] preprints); lipid organization (e.g., microdomain association of LAT [28] and viral glycoproteins [27]); complex formation (e.g., ankyrin-G, GPCRs [20,21]); protein trafficking (e.g., Ras, integral membrane proteins [25,38,39]); signal transduction (NOD2 and Myd88 [17][18][19]); and crosstalk with other post-translational modifications (PTMs) (e.g., tyrosinase, BK potassium channels [30,31]). Cartoons were generated with Biorender.com poorly characterized acyl-protein thioesterases (APTs), members of the serine hydrolase superfamily [29].…”

“…2. These include modulation of protein conformation [12][13][14][15][16]; capacity to transduce signals [17][18][19]; ability to interact with other proteins [18,20,21]; regulation of their trafficking and localization [12,[22][23][24][25]; physicochemical properties of their transmembrane domains (TMDs) and their association with surrounding lipids [26][27][28]; and cross-talk with other PTMs [29][30][31]. By modifying such a vast number of proteins and affecting their function through a broad diversity of means, S-acylation is involved in various diseases, including neurodegenerative diseases, cancer, and inflammatory diseases (reviewed in [10,19,32]).…”

“…Consequences of protein S-acylation. Schematic representation of selected potential consequences of S-acylation on cytosolic and membranes proteins depicting examples of membrane association (e.g., Src kinases[33]); conformational changes (e.g., voltage-gated channels[14][15][16]); multimerization (e.g., STING[34,35], Gasdermin D pores[36,37] preprints); lipid organization (e.g., microdomain association of LAT[28] and viral glycoproteins[27]); complex formation (e.g., ankyrin-G, GPCRs[20,21]); protein trafficking (e.g., Ras, integral membrane proteins[25,38,39]); signal transduction (NOD2 and Myd88[17][18][19]); and crosstalk with other post-translational modifications (PTMs) (e.g., tyrosinase, BK potassium channels[30,31]). Cartoons were generated with Biorender.com…”

Palmitoylation regulates the magnitude of HCN4-mediated currents in mammalian cells

“…In recent years, palmitoylation has emerged as an important post translational modification regulating many physiological and pathophysiological processes in the heart ( Essandoh et al, 2020 ; Main et al, 2022 ; Main and Fuller, 2022 ). Essential cardiac sodium ( Reilly et al, 2015 ; Pei et al, 2016 ; Gok et al, 2020 ; Gok et al, 2022 ) and calcium ( Kuo et al, 2023 ) handling proteins are dynamically palmitoylated, influencing their membrane microdomain localisation and function. Palmitoylation regulates the activities of several proteins known to be central to SAN pacemaking, including NCX1 ( Reilly et al, 2015 ; Gok et al, 2020 ) and Ca(v)1.2, 25 and all isoforms of HCN channels except HCN3 are palmitoylated ( Itoh et al, 2016 ).…”