title: Palmitoylation and regulation of the funny current HCN4 channel
Creators: name: Congreve, Samitha Dilini
Publisher: name: University of Glasgow
Year: 2023

description: The sinoatrial node (SAN) acts as the primary pacemaker of the heart as it spontaneously generates electrical activity that propagate through the cardiac conduction system, underpinning automaticity of the heart. A network of surface membrane ion currents (“membrane clock”) and the rhythmic oscillation of local Ca²⁺ release from the sarcoplasmic reticulum (“calcium clock”) work interdependently to form a coupled-clock system that drives pacemaker automaticity and its regulation on a beat-to-beat basis. The “funny current” (If) is a key component of the membrane clock contributing to the diastolic depolarisation of the SAN. Hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is the predominant isoform responsible for almost 70% of the sinoatrial If. HCN4 channels localise to lipid rafts in the SAN and disorganisation of these raft membrane microdomains result in channel redistribution, thus altering its kinetic properties. Ion channels are an integral component of the complex sinoatrial pacemaking network, and their regulation is therefore central to controlling the heart rate. S-palmitoylation is a form of lipidation that involves the covalent addition of a 16-carbon palmitate to a thiol group of a cysteine residue in a protein. Unlike most lipid modifications, palmitoylation is unique due to its reversible nature, allowing the dynamic regulation of both soluble and integral proteins. In recent years, palmitoylation has emerged as an important regulator of cardiac electrophysiology as it influences the function and membrane microdomain localisation of key cardiac Na⁺ and Ca²⁺ handling proteins. The present in-vitro study was adopted to characterise palmitoylation of HCN4 channels and to establish its functional consequences. Site-specific resin assisted capture (acyl-RAC) was used to assess palmitoylation of HCN4 in human embryonic kidney (HEK) cells as well as endogenous HCN4 in isolated neonatal rat whole heart and atrial myocytes. HCN4 was sub-stoichiometrically palmitoylated in all experimental systems examined. Truncated HCN4 intracellular amino and carboxyl termini fused to YFP and cysteine-to-alanine mutations of the palmitoylation sites in HEK-293 cells mapped HCN4 palmitoylation sites to a pair of cysteines (C93 and C179) in the HCN4 N-terminus domain. A double cysteine-to-alanine mutation C93/179AA of both palmitoylation sites reduced palmitoylation of full-length HCN4 by ~67% in comparison to wild type HCN4. Membrane impermeable biotinylation of cell surface HCN4 revealed that palmitoylation did not influence its trafficking to the cell surface or cell surface turnover rate. Standard discontinuous sucrose gradient indicated that HCN4 channels did not require palmitoylation to localise to lipid rafts in HEK-293 cells. Whole-cell patch clamp was used to investigate IHCN4 in HEK-293 cells engineered to stably express wild type and mutant HCN4. Loss of palmitoylation at the N-terminus significantly reduced HCN4 current magnitude by ~5 to 8-fold across a range of voltages. However, it did not alter its half-maximal activation voltage (V₀.₅: -90.4 ± 2.5 mV for WT vs -90.4 ± 1.6 mV for C93/179AA), nor its activation slope factor (k: 7.1 ± 0.5 mV for WT vs 6.0 ± 0.2 mV for C93/179AA). Phylogenetic analysis was used to evaluate the evolutionary acquisition of HCN4 palmitoylation within the pre-metazoan and metazoan lineage. While cysteine 93 was broadly conserved within all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 was confined to placental mammals. Together, this study demonstrated the importance of palmitoylation as a regulator of HCN4 channel function by enhancing HCN4-mediated currents. Palmitoylation of the HCN4 amino terminus is likely to significantly enhance If in the SAN, accelerating diastolic depolarisation, and increasing heart rate.