{'fraction_non_alphanumeric': 0.03108350793175696, 'fraction_numerical': 0.018901526489075126, 'mean_word_length': 5.342762221167537, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 4, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Ion channel regulation by protein S-acylation

Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease.

and various mammalian membrane proteins (Schmidt and Schlesinger, 1979;Schlesinger et al., 1980). A decade later, Sacylated ion channels-rodent voltagegated so dium channels (Schmidt and Catterall, 1987) and the M2 ion channel from the influenza virus (Sugrue et al., 1990)-were first characterized. Since then, more than 50 distinct ion channel subunits have been experimen tally demonstrated to be Sacylated (Tables 1-3) as have a wide array of structural, signaling, and scaffolding proteins (for reviews see Linder and Deschenes, 2007;Fukata and Fukata, 2010;Greaves and Chamberlain, 2011;Resh, 2012). In the last few years, with the cloning of enzymes controlling Sacylation and development of various proteomic tools, we have begun to gain substantial mechanistic and physi ological insight into how Sacylation may control mul tiple facets of the life cycle of ion channels: from their assembly, through their trafficking and regulation at the plasma membrane, to their final degradation (Fig. 2).
Here, I provide a primer on the fundamentals of Sacylation, in the context of ion channel regulation, along with a brief overview of tools available to interro gate ion channel Sacylation. I will discuss key examples of how Sacylation controls distinct stages of the ion channel life cycle before highlighting some of the key challenges for the field in the future.

Ion channel regulation by protein S-acylation
Michael J. Shipston Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH8 9XD Scotland, UK Protein Sacylation, the reversible covalent fattyacid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numer ous ligand and voltagegated ion channels. Sacylation is enzymatically mediated by a diverse family of acyltrans ferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which Sacylation and deacylation cycles occur are not known. Sacylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modu lated by Sacylation of both poreforming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, crosstalk of Sacylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein Sacylation and the tools available to investigate ion channel Sacylation. The mechanisms and role of Sacylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for Sacylation control of ion channels and the functional consequence and im plications for understanding the physiological function of ion channel Sacylation in health and disease.

Fundamentals of S-acylation:
The what, when, where, and how S-acylation: A fatty modification that controls multiple aspects of protein function. Protein Sacylation results from the attachment of a fatty acid to intracellular cysteine residues of proteins via a labile, thioester linkage (Fig. 1,  A and B). Because the thioester bond is subject to nu cleophilic attack, Sacylation, unlike other lipid modi fications such as Nmyristoylation and prenylation, is reversible. However, for most ion channels, as for other Sacylated proteins, the dynamics of Sacylation are poorly understood. Distinct classes of proteins can un dergo cycles of acylation and deacylation that are very rapid (e.g., on the timescale of seconds, as exemplified by rat sarcoma [RAS] proteins), much longer (hours), or essentially irreversible during the lifespan of the protein Linder and Deschenes, 2007;Zeidman et al., 2009;Fukata and Fukata, 2010;Greaves and Chamberlain, 2011;Resh, 2012). For most ion channels, in fact most Sacylated proteins, the iden tity of the native lipid species attached to specific cys teine residues is also largely unknown. However, the saturated C16:0 lipid palmitate is commonly thought to be the major lipid species in many Sacylated proteins (Fig. 1). Indeed, much of the earliest work on Sacyl ation involved the metabolic labeling of proteins in cells with tritiated [ 3 H]palmitate, an approach that still re mains useful and important. However, lipids with dif ferent chain lengths and degrees of unsaturation (such as oleic and stearic acids) can also be added to cysteines via a thioester linkage, potentially allowing differential control of protein properties through the attachment of distinct fatty acids Linder and Deschenes, 2007;Zeidman et al., 2009;  Sacylation is reversible due to the labile thioester bond between the lipid (typically, but not exclusively, palmitate) and the cysteine amino acid of is target protein. Other lipid modifications result from stable bond formation between either the Nterminal amino acid (amide) or the amino acid side chain in the protein (thioether and oxyester). The zDHHC family of palmitoyl acyltransferases medi ates Sacylation with other enzyme families controlling other lipid modifications: Nmethyltransferase (NMT) controls myristoylation of many proteins such as the src family kinase, Fyn kinase; and amidelinked palmitoylation of the secreted sonic hedgehog protein is mediated by Hedgehog acyltransferase (Hhat), a membranebound Oacyl transferase (MBOAT) family. Prenyl transferases catalyze farnesyl (farnesyltransferase, FTase) or geranylgeranyl (geranylgeranyl transferase I [GGTase I] and geranylgeranyl transferase II [GG Tase II]) in small GTPase proteins such as RAS and the Rab proteins, respectively. Porcupine (Porcn) is a member of the MBOAT family acylates secreted proteins such as Wnt. (B) zDHHC enzymes typically use coenzyme A (CoA)palmitate; however, other long chain fatty acids (either saturated or desaturated) can also be used. Deacylation is mediated by several acylthioesterases of the serine hydrolase family. (C) zDHHC acyltransferases (23 in humans) are predicted transmembrane proteins (typically with 4 or 6 transmembrane domains) with the catalytic DHHC domain located in a cytosolic loop.   Linder and Deschenes, 2007), most cellular Sacylation, in organisms from yeast to humans, is thought to be en zymatically driven by a family of protein acyltransferases (gene family: zDHHC, with 23 members in mammals). These acyltransferases are predicted to be transmem brane zinc finger containing proteins ( Fig. 1 C) that include a conserved AspHisHisCys (DHHC) signature sequence within a cysteinerich stretch of 50 amino acids critical for catalytic activity (Fukata et al., 2004). Although the enzymatic activity and lipid specificity of all of the zDHHC family proteins has not been elucidated, Sacylation is thought to proceed through a common, two step "ping pong" process (Mitchell et al., 2010;Jennings and Linder, 2012). However, different zDHHC enzymes may show different acylCoA substrate specificities. For example, zDHHC3 activity is reduced by acyl chains of >16 carbons (e.g., stearoyl CoA), whereas zDHHC2 ef ficiently transfers acyl chains of 14 carbons or longer (Jennings and Linder, 2012). The local availability of different acylCoA species may thus play an important role in differentially controlling protein Sacylation. and Fukata, 2010;Greaves and Chamberlain, 2011;Resh, 2012). Sacylation increases protein hydrophobicity and has thus been implicated in controlling protein function in many different ways. Most commonly, as with mem braneassociated proteins like RAS and postsynaptic density protein 95 (PSD95), Sacylation controls mem brane attachment and intracellular trafficking. However, Sacylation can also control protein-protein interactions, protein targeting to membrane subdomains, protein sta bility, and regulation by other PTMs such as phosphory lation Fukata and Fukata, 2010;Linder and Deschenes, 2007;Greaves and Cham berlain, 2011;Shipston, 2011;Resh, 2012). Evidence for all these mechanisms in controlling ion channel func tion is beginning to emerge.

Enzymatic control of S-acylation by zinc finger-containing acyltransferase (zDHHC) transmembrane acyltransferases.
Although autoacylation of some proteins has been re ported in the presence of acyl coenzyme A (acylCoA; T a b l E 2 Common channel abbreviation and subunit as well as gene names are given. Candidate Sacylation sites: experimentally determined cysteine residues (bold) with flanking 10 amino acids. Underlines indicate predicted transmembrane domains. Amino acid numbering corresponds to the UniProt ID. References: selected original supporting citations.

Accessory subunits and selected ion channel adapter proteins
T a b l E 1 (Continued) by distinct subsets of zDHHCs . Although we are still in the foothills of understanding the substrates and physiological roles of different zDHHCs, mutation or loss of function in zDHHCs is associated with an increasing number of human disorders, including cancers, various neurological disorders (such as Hun tington's disease and Xlinked mental retardations), and disruption of endocrine function in diabetes (Linder and Deschenes, 2007;Fukata and Fukata, 2010;Greaves and Chamberlain, 2011;Resh, 2012).
Deacylation is controlled by acylthioesterases. Protein de acylation is enzymatically driven by a family of acylthioes terases that belong to the serine hydrolase superfamily (Zeidman et al., 2009;Bachovchin et al., 2010). Indeed, using a broad spectrum serine lipase inhibitor, global proteomic Sacylation profiling identified a subset of ser ine hydrolases responsible for depalmitoylation . This study identified both the previously known acylthioesterases as well as potential novel candi date acylthioesterases. The acylthioesterases responsible We know very little about how zDHHC activity and function are regulated. Dimerization of zDHHCs 2 and 3 reduces their zDHHC activity compared with the mono meric form (Lai and Linder, 2013). Moreover, zDHHCs undergo autoacylation and contain predicted sites for other posttranslational modifications. Almost half of all mammalian zDHHCs contain a Cterminal PSD95, Discs large, and ZO1 (PDZ) domain binding motif, allowing them to assemble with various PDZ domain proteins that regulate ion channels (such as GRIP1b and PSD95; . Other protein interaction domains are also observed in zDHHCs, such as ankyrin repeats in zDHHC17 and zDHHC13 (Greaves and Chamberlain, 2011). Indeed, increasing evidence suggests that various ion channels-including the ligandgated amino butyric (GABA A ), amino3hydroxyl5methyl4isoxazolepro pionate (AMPA), and NMDA receptors and the large conductance calcium and voltageactivated (BK) potas sium channels-can assemble in complexes with their cognate zDHHCs.
The expansion of the number of zDHHCs in mam mals (23 vs. 7 in yeast), together with increased prev alence of PDZ interaction motifs, likely represents evolutionary gainoffunction mechanisms to diversify zDHHC function . Evo lutionary gain of function is also seen in ion channel subunit orthologues through acquisition of Sacylated cysteine residues absent in orthologues lower in the phy logenetic tree (such as the transmembrane domain 4 [TM4] sites in GluA1-4 subunits of AMPA receptors  and the sites in the alter natively spliced stressregulated exon [STREX] insert in the C terminus of the BK channel [Tian et al., 2008]). Importantly, some zDHHCs may have additional roles beyond their acyltransferase function. For example, the Drosophila melanogaster zDHHC23 orthologue lacks the catalytic DHHC sequence, and thus protein acyltrans ferase activity, and is a chaperone involved in protein trafficking (Johswich et al., 2009), whereas mammalian zDHHC 23 has a functional zDHHC motif and, in addi tion to Sacylating BK channels , can bind and regulate, but does not Sacylate, neuronal ni tric oxide synthase (nNOS; Saitoh et al., 2004).
However, as with most Sacylated proteins, the identity of the zDHHCs that modify specific cysteine residues on individual ion channels is not known. Indeed, rela tively few studies have tried to systematically identify the zDHHCs controlling ion channel function . Thus we are largely ignorant of the extent to which different zDHHCs may have specific ion chan nel targets or may display specificity. Some details are beginning to emerge: for example, zDHHC3 appears to be a rather promiscuous acyltransferase reported to Sacylate several ion channels (Keller et al., 2004;Hayashi et al., 2005Hayashi et al., , 2009Tian et al., 2010), whereas distinct sites on the same ion channel subunit can be modified

Other channels identified in mammalian palmitoylome screens
Channel Gene
S-acylation occurs at membrane interfaces. Because the zDHHCs are transmembrane proteins and the catalytic DHHC domain is located at the cytosolic interface with membranes ( Fig. 1 C), Sacylation of ion channels oc curs at membrane interfaces. Although overexpression studies of recombinant mammalian zDHHCs in heter ologous expression systems have indicated that most zDHHCs are localized to either the endoplasmic reticu lar or Golgi apparatus membranes (or both; Ohno et al., 2006), some zDHHCs are also found in other compart ments, including the plasma membrane and trafficking endosomes (Thomas et al., 2012;Fukata et al., 2013).
We know very little about the regulation and subcellular localization of most native zDHHC enzymes in different cell types, in large part because of the lack of highqual ity antibodies that recognize native zDHHCs. However, some enzymes, including zDHHC2, can dynamically shuttle between different membrane compartments. Activitydependent redistribution of zDHHC2 in neurons (Noritake et al., 2009) controls Sacylation of the post synaptic scaffolding protein PSD95, thereby regulating NMDA receptor function. Intriguingly, as ion channels themselves determine cellular excitability, this may pro vide a local feedback mechanism to regulate Sacylation status. Thus, although different zDHHCs may reside in multiple membrane compartments through which ion channels traffic, the subcellular location at which most ion channels are Sacylated, as well as the temporal dy namics, is largely unknown. As discussed below (see the "Tools to analyze ion channel Sacylation" section), we are starting to unravel some of the details, with ER exit, Golgi retention, recycling endosomes, and local plasma membrane compartments being key sites in the control of ion channel Sacylation (Fig. 2).
Local membrane and protein environment determines cysteine S-acylation. The efficiency of Sacylation of cysteine residues is likely enhanced by its localization at mem branes because the local concentration of fatty acyl CoA is increased near hydrophobic environments (Bélanger et al., 2001). Furthermore, Sacylation of polytopic trans membrane proteins such as ion channels would be fa cilitated when Sacylated cysteines are bought into close proximity of membranes by membrane targeting mech anisms such as transmembrane helices (Figs. 3 and 4). However, the Sacylated cysteine is located within 10 amino acids of a transmembrane domain in only 20% of identified Sacylated ion channel subunits, such as the TM4 site of GluA1-4 (Tables 1 and 2). Most Sacylated cysteines are located either within intracellular loops (40%: Fig. 3, A and B) or the N or Cterminal cy tosolic domains (5% and 35%, respectively; Fig. 3, A and B). Furthermore, the majority of Sacylated cys teines located in intracellular loops or intracellular N or Cterminal domains of ion channel subunits are within predicted regions of protein disorder (Fig. 3 B).
for deacylating ion channels, as for most other acylated membrane proteins, have not been clearly defined. Fur thermore, the extent to which different members of the serine hydrolase superfamily display acylthioesterase activity toward ion channels is not known. Moreover, whether additional mechanisms of nucleophilic attack of the labile thioester bond may also mediate deacyla tion is not known.
Homeostatic control of deacylation of many signal ing proteins is likely affected by a family of cytosolic acyl protein thioesterases including lysophospholipase 1 (LYPLA1; Yeh et al., 1999;Devedjiev et al., 2000) and ly sophospholipase 2 (LYPLA2; Tomatis et al., 2010). These enzymes show some selectivity for different Sacylated peptides (Tomatis et al., 2010). Indeed, LYPLA1, but not LYPLA2, deacylates the S0S1 loop of BK channels, lead ing to Golgi retention of the channel . A splice variant of the related LYPLAL1 acylthioesterases can also deacylate the BK channel S0S1 loop, although the crystal structure of LYPLAL1 suggests it is likely to have a preference for lipids with shorter chains than pal mitate (Bürger et al., 2012). Thus, whether lipid prefer ence depends on protein interactions or if BK channels have multiple lipid species at the multicysteine S0S1 site remain unknown. Relatively little is known about the reg ulation of these acylthioesterases; however, both LYPLA1 and LYPLA2 are themselves Sacylated. This controls their trafficking and association with membranes (Kong et al., 2013;Vartak et al., 2014) and may be important for accessing the thioesterase bond at the membrane inter face. Additional mechanisms may promote accessibility of thioesterases to target cysteines. For example, the prolyl isomerase protein FKBP12 binds to palmitoylated RAS, and promotes RAS deacylation via a proline residue near the Sacylated cysteine (Ahearn et al., 2011).
Upon lysosomal degradation, many proteins are de acylated by the lysosomal palmitoyl protein thioesterase (PPT1; Verkruyse and Hofmann, 1996), and mutations in PPT1 lead to the devastating condition of infantile neuronal ceroid lipofuscinosis (Vesa et al., 1995;Sarkar et al., 2013). However, PPT1 can also be found in synap tic and other transport vesicles, and genetic deletion of PPT1 in mice may have different effects on similar pro teins, which suggests roles beyond just lysosomal medi ated degradation. For example, in PPT1 knockout mice the total expression and surface membrane abundance of the GluA4 AMPA receptor subunit was decreased, whereas PPT1 knockout had no effect on GluA1 or GluA2 AMPA subunits nor on NMDA receptor subunit expres sion or surface abundance (Finn et al., 2012).
However, for most ion channels, the questions of which enzymes control deacylation, where this occurs in cells, and how the time course of acylation-deacylation cycles are regulated are largely unknown. Thus, whether deacyla tion plays an active role in channel regulation remains poorly understood. proximity to basic (or hydrophobic) residues (Bélanger et al., 2001;Britto et al., 2002;Kümmel et al., 2010). Fur thermore, cysteine residues are subject to a range of modifications including nitrosylation, sulphydration, reductionoxidation (REDOX) modification, and for mation of disulphide bonds (Sen and Snyder, 2010). Evidence is beginning to emerge that these reversible modifications are mutually competitive for Sacylation of target cysteines (see the "Sacylation and posttransla tional crosstalk controls channel trafficking and activ ity" section; Ho et al., 2011;Burgoyne et al., 2012).
Although these linear amino acid sequence features are likely to be important for efficient Sacylation, there is no canonical "consensus" Sacylation motif analogous to the linear amino acid sequences that predict sites of phosphorylation. Of the experimentally validated ion channel subunits shown to be Sacylated, 70% of can didate Sacylated cysteines are predominantly charac terized as single cysteine (C) motifs, whereas dicysteine motifs (CC) and (CX (1-3) C) motifs comprise 10% and 20% of all sites, respectively (Table 1 and Fig. 3 B). However, several freely available online predictive tools have proved successful in characterizing potential new palmitoylation targets. In particular, the latest itera tion of the multiplatform CSSpalm 4.0 tool (Ren et al., 2008) exploits a Groupbased prediction algorithm by comparing the surrounding amino acid sequence simi larity to that of a set of 583 experimentally determined This suggests that Sacylation may provide a signal to promote conformational restraints on such domains, in particular by providing a membrane anchor. For these sites, additional initiating membrane association signals are likely required adjacent to the site of Sacylation. Likely candidates include other hydrophobic domains (as for the TM2 site in GluA1-4 subunits; Fig. 4 A) and other lipid anchors (e.g., myristoylation in src family ki nases, such as Fyn kinase). However, in >30% of Sacyl ated ion channels, the Sacylated cysteine is juxtaposed to a (poly) basic region of amino acids that likely allows electrostatic interaction with negative membrane phos pholipids. The BK channel poreforming  subunit, encoded by the KCNMA1 gene, provides a clear exam ple of this latter mechanism. This channel is Sacylated within an alternatively spliced domain (STREX) in its large intracellular C terminus (Fig. 4 C). Immediately upstream of the Sacylated dicysteine motif is a poly basic region enriched with arginine and lysine. Site directed mutation of these basic amino acids disrupts Sacylation of the downstream cysteine residues (Jeffries et al., 2012). Furthermore, phosphorylation of a con sensus PKA site (i.e., introduction of negatively charged phosphate) into the polybasic domain prevents STREX Sacylation. Thus, at the STREX domain, an electro static switch, controlled by phosphorylation, is an im portant determinant of BK channel Sacylation. In other proteins, cysteine reactivity is also enhanced by interaction with the actinbinding protein 4.1N in neu rons, leading to enhanced AMPA plasma membrane in sertion (Lin et al., 2009). Sacylation of the Cterminal cluster of cysteine residues (Fig. 4 B, Cys II site) in GluN2A and GluN2B controls Golgi retention, whereas palmitoylation of the cysteine cluster (Cys I site) proxi mal to the M4 transmembrane domain controls channel internalization . Distinct roles of Sacylation on channel trafficking and regulation are also observed in BK channels (Figs. 4 C and 5). Sacylation of the Nterminal intracellular S0S1 linker controls sur face expression, in part by controlling ER and Golgi exit of the channel Tian et al., 2012), whereas Sacylation of the large intracellular C terminus, within the alternatively spliced STREX domain, controls BK channel regulation by AGC family protein kinases (Tian et al., 2008;Zhou et al., 2012).
How does Sacylation of distinct domains control such behavior, and are distinct sites on the same protein acyl ated by distinct zDHHCs? A systematic small interfer ing RNA (siRNA) screen of zDHHC enzymes mediating BK channel Sacylation indicated that distinct subsets of zDHHCs modify discrete sites. The S0S1 loop is Sacyl ated by zDHHCs 22 and 23, whereas the STREX domain is Sacylated by several zDHHCs including 3, 9, and 17 (Tian et al., 2008. In both cases, each domain has two distinct Sacylated cysteines; however, whether these cysteines are differentially Sacylated by specific zDHHCs is unknown, Furthermore, whether multiple zDHHCs are required because the domains undergo repeated cycles of Sacylation and deacylation, and thus Sacylation sites from 277 distinct proteins. CSSpalm 4.0 predicts >80% of the experimentally identified ion channel Sacylation sites (Tables 1-3) and suggests that >50% of human channel subunits may be Sacylated.
Location of S-acylated cysteine is important for differential control of channel function. Many proteins are Sacyl ated at multiple sites. A remarkable example of this, in the ion channel field, is the recent identification of 18 Sacyl ated cysteine residues in the skeletal muscle ryanodine receptor/Ca 2+ release channel (RyR1). The Sacylated cysteine residues are distributed throughout the cytosolic N terminus, including domains important for proteinprotein interactions (Chaube et al., 2014). Although deacylation of skeletal muscle RyR1 reduces RyR1 activ ity, the question of which of these cysteine residues in RyR1 are important for this effect and whether distinct Sacylated cysteines in RyR1 control different functions and/or properties remains to be determined.
However, both ligandgated (NMDA and AMPA) and voltagegated (BK) channels provide remarkable insights into how Sacylation of different domains within the same polytopic protein can exert fundamentally distinct effects (Fig. 4). For example, Sacylation of the hydrophobic cy tosolic TM2 domain located at the membrane interface of the AMPA GluA1 subunit (Fig. 4 A) decreases AMPA receptor surface expression by retaining the subunit at the Golgi apparatus (Hayashi et al., 2005). In contrast, depalmitoylation of the Cterminal cysteine in GluA1 results in enhanced PKCdependent phosphorylation of neighboring serine residues, which results in increased phosphorylation even though a PKC phosphorylation site serine motif, conserved in other BK channel vari ants, is present downstream of the STREX domain. In other BK channel variants lacking the STREX insert, this PKC site is required for channel inhibition by PKCdependent phosphorylation. However, after deac ylation of the STREX domain, PKC can now phosphor ylate this PKC phosphorylation serine motif, which suggests that the site has become accessible, conse quently resulting in channel inhibition (Fig. 5;Zhou et al., 2012).
How might Sacylation of a cysteine residue juxta posed to another membrane anchoring domain control protein function? The simplest mechanism would in volve acting as an additional anchor (Fig. 3 A). In some systems, juxtatransmembrane palmitoylation allows tilting of transmembrane domains, effectively shorten ing the transmembrane domain to reduce hydrophobic mismatch (Nyholm et al., 2007), particularly at the thin ner ER membrane (Abrami et al., 2008;Charollais and Van Der Goot, 2009;Baekkeskov and Kanaani, 2009), and confer conformational restraints on the peptide (Fig. 3 A). Such a mechanism has been proposed to con trol ER exit of the regulatory 4 subunits of BK chan nels. In this case, depalmitoylation of a cysteine residue juxtaposed to the second transmembrane domain of the 4 subunits may result in hydrophobic mismatch at the ER, reducing ER exit, and yield a conformation that is unfavorable for interaction with BK channel  subunits, thereby decreasing surface expression of BK channel  subunits (Chen et al., 2013). different zDHHCs function at different stages of the protein lifecycle, remains to be determined. Although systematic siRNA screens have, to date, not been per formed on other ion channels, data from other multi ply Sacylated channels, such as NMDA, AMPA, and BK channel subunits, supports the hypothesis that zDHHCs can show substrate specificity (Hayashi et al., 2005Tian et al., 2010).
It is generally assumed that Sacylation facilitates the membrane association of protein domains. This is clearly the case for peripheral membrane proteins, such as RAS or PSD95, but direct experimental evidence for Sacylation controlling membrane association of the cy tosolic domains of transmembrane proteins is largely elusive. One of the best examples involves the large Cterminal domain of the BK channel, which com prises more than twothirds of the poreforming subunit (Fig. 5). In the absence of Sacylation of the STREX do main, or exclusion of the 59-amino acid STREX insert, the BK channel C terminus is cytosolic (Tian et al., 2008). However, if the STREX domain is Sacylated, the en tire C terminus associates with the plasma membrane, a process that can be dynamically regulated by phos phorylation of a serine immediately upstream of the Sacylated cysteines in the STREX domain (Tian et al., 2008). This Sacylation-dependent membrane associa tion markedly affects the properties and regulation of the channel (Jeffries et al., 2012) and has been pro posed to confer significant structural rearrangements. In support of such structural rearrangement, Sacylated STREX channels are not inhibited by PKCdependent an in silico platform to inform experimental approaches for candidate targets.
Pharmacological tools. The Sacylation pharmacologi cal toolkit remains, unfortunately, empty, with limited specific agents with which to explore Sacylation func tion in vitro or in vivo. Although the palmitate analogue 2bromopalmitate (2BP) is widely used for cellular as says and to analyze ion channel regulation by Sacylation, caution must be taken in using this agent, even though it remains our best pharmacological inhibitor of zDHHCs (Resh, 2006;Davda et al., 2013;Zheng et al., 2013). Un fortunately, 2BP is a nonselective inhibitor of lipid me tabolism and many membraneassociated enzymes, and displays widespread promiscuity (e.g., Davda et al., 2013); does not show selectivity toward specific zDHHC pro teins (Jennings et al., 2009); has many pleiotropic effects

Tools to analyze ion channel S-acylation
Before the seminal discovery of the mammalian enzymes that control Sacylation (Fukata et al., 2004) and current advances in proteomic techniques to assay Sacylation, progress in the field was relatively slow, largely because of the lack of pharmacological, proteomic, and genetic tools to investigate the functional role of Sacyl ation. It is perhaps instructive to consider that protein tyrosine phosphorylation was discovered the same year as Sac ylation (Hunter, 2009). However, the subsequent rapid identification and cloning of tyrosine kinases provided a very extensive toolkit to investigate this pathway. Al though the Sacylation toolkit remains limited, the last few years have seen rapid progress in our ability to inter rogate Sacylation function and its control of ion channel physiology. Furthermore, Sacylation prediction algo rithms, such as CSSpalm 4.0 (Ren et al., 2008), provide Figure 5. Sacylation controls BK channel trafficking and regulation by AGC family protein kinases via distinct sites. The BK channel STREX splice variant poreforming  subunit is Sacylated at two sites: the S0S1 loop and the STREX domain in the large intracellular C terminus. Sacylation of the S0S1 loop promotes high surface membrane expression of the channel; thus, deacylation of this site de creases the number of channels at the cell surface (see the "Control of ion channel cell surface expression and spatial organization in membranes" section for further details). In contrast, Sacylation of the STREX domain allows inhibition of channel activity by PKAme diated phosphorylation of a PKA serine motif (closed hexagon) immediately upstream of the palmitoylated cysteine residues in STREX. In the Sacylated state, PKC has no effect on channel activity even though a PKC phosphorylation site serine motif is located immediately downstream of the STREX domain (open triangle). Deacylation of STREX dissociates the STREX domain from the plasma membrane, and exposes the PKC serine motif so that it can now be phosphorylated by PKC (closed triangle), resulting in channel inhibition. In the deacylated state, PKA has no effect on channel activity (open hexagon). Thus, deacylation of the STREX domain switches channel regulation from a PKAinhibited to a PKCinhibited phenotype (see the "Sacylation and posttranslational crosstalk controls channel trafficking and activity" section for further details). or knocking down individual zDHHCs on the localiza tion and activity of other zDHHCs must also be carefully determined. For example, siRNAmediated knockdown of zDHHC 5, 7, or 17 in HEK293 cells paradoxically re sults in an upregulation of zDHHC23 mRNA expression . Furthermore, because many signaling and cytoskeletal elements are also controlled by Sacyla tion, direct effects on channel Sacylation by themselves must be evaluated in parallel (for example using sitedi rected cysteine mutants of the channel subunit). Fewer studies have used these approaches to examine the role of acylthioesterases, although overexpression of LYPLA1 and a splice variant of LYPLAL1, but not LYPLA2, deacyl ates the S0S1 loop of the BK channel, promoting Golgi retention of the channels . Genetrap and knockout mouse models for some zDHHCs (such as 5 and 17) are becoming available, although full phe notypic analysis and analysis of ion channel function in these models are largely lacking.
Proteomic and imaging tools. Lipid-centric (metabolic) labeling assays. Metabolic labeling approaches are most suited to analysis of isolated cells, rather than tissues, but pro vide information on dynamic palmitoylation of proteins during the relatively short (4 h) labeling period as well as insight into the species of lipid bound to cysteine resi dues. The classical approach using radioactive palmitate (e.g., [ 3 H]palmitate) remains a "gold standard" for valida tion, in particular for identification that palmitate is the bound lipid. However, metabolic labeling with [ 3 H]pal mitate generally requires immunoprecipitation and days to weeks of autoradiography or fluorography, particu larly when analyzing low abundance membrane proteins such as ion channels. To overcome some of these issues, and also to provide a platform to allow cellular imaging of Sacylation, a variety of biorthogonal lipid probes have recently been developed (Hannoush and ArenasRamirez, 2009;Hannoush, 2012;Martin et al., 2012; for reviews see Charron et al., 2009a;Hannoush and Sun, 2010). These probes are modified fatty acids with reactive groups, such as an azide or alkyne group, allowing labeled proteins to be conjugated to biotin or fluorophores via the reactive group using Staudinger ligation or "click" chemistry. In particular, development of a family of alkynyl fatty acid probes of different chain lengths (such as AlkC16 and AlkC18) have been exploited for proteomic profiling as well as single cell imaging (Gao and Hannoush, 2014) and have been used to identify candidate Sacylated chan nels in several mammalian cell lines (Table 3; Charron et al., 2009b;Hannoush and ArenasRamirez, 2009;Martin and Cravatt, 2009;Yap et al., 2010;Yount et al., 2010;Martin et al., 2012). It is important to note that pal mitic acid can also be incorporated into free Nterminal cys teines of proteins via an amide linkage (Npalmitoylation), addition of the monounsaturated palmitoleic acid via an oxyester linkage to a serine residue (Opalmitoylation), on cells at high concentrations, including cytotoxicity (Resh, 2006); and also inhibits acylthioesterases (Pedro et al., 2013). Other lipid inhibitors include cerulenin and tunicamycin. However, cerulenin affects many aspects of lipid metabolism, and tunicamycin inhibits Nlinked glycosylation (Resh, 2006). Although some nonlipid in hibitors have been developed, these are not widely used (Ducker et al., 2006;Jennings et al., 2009), and there are currently no known activators of zDHHCs or compounds that inhibit specific zDHHCs. In the last few years, several inhibitors for the acylthioesterases LYPLA1 and LYPLA2 have been developed (Bachovchin et al., 2010;Dekker et al., 2010;Adibekian et al., 2012). However, several of these compounds, such as palmostatin B, are active against several members of the larger serine hydrolase family. Clearly, the development of novel Sacylation in hibitors and activators that display both specificity and zDHHC selectivity would represent a substantial advance for investigation of channel Sacylation.
Genetic tools. To date, most studies have used overexpres sion of candidate zDHHCs in heterologous expression or native systems and analyzed increases in [ 3 H]palmitate incorporation to define zDHHCs that may Sacylate spe cific ion channels (e.g. Rathenberg et al., 2004;Hayashi et al., 2005Hayashi et al., , 2009Tian et al., 2010;Thomas et al., 2012). Although this is a powerful approach, caution is required to determine whether results obtained with overexpres sion in fact replicate endogenous regulation. For exam ple, overexpression of some zDHHCs normally expressed in the cell type of interest can result in Sacylation of a cysteine residue that is not endogenously palmitoylated in BK channels . Point mutation of the cysteine of the catalytic DHHC domain abolishes the acyl transferase activity of zDHHCs and is thus an invaluable approach to confirming that the acyltransferase function of overexpressed zDHHC is required by itself. Increas ingly, knockdown of endogenous zDHHCs using siRNA, and related approaches, is beginning to reveal the iden tity of zDHHCs that Sacylate native ion channel subunits. For example, knockdown of zDHHCs 5 or 8 reduces Sacyl ation of the accessory subunits PICK1 and Grip1, which control AMPA receptor trafficking (Thomas et al., 2012; and knockdown of zDHHC2 disrupts local nano clusters of the PDZ domain protein PSD95 in neuronal dendrites to control AMPA receptor membrane localiza tion (Fukata et al., 2013). However, relatively few studies have taken a systematic knockdown approach to identify zDHHCs important for ion channel Sacylation. One such approach has, however, revealed that multiple, distinct zDHHCs mediate palmitoylation of the BK channel C terminus (zDHHCs 3,5,7,9,and 17) and that a differ ent subset of zDHHCs (22 and 23) mediate Sacylation of the intracellular S0S1 loop in the same channel . Because some zDHHCs are themselves palmitoylated, the functional effect of overexpressing phosphospecific antibodies to analyze phosphoproteins in cells and tissues) are largely lacking in the Sacylation field. As a result, for example, spatial analysis of native Sacylation in cells or tissue sections is largely lacking. Recently, an intrabody that recognizes the conforma tional rearrangement of the ion channel scaffolding protein PSD95 upon Sacylation, allowing cycles of Sacylation and deacylation of native PSD95 to be mon itored in neurons, has recently been developed (Fukata et al., 2013). However, this approach does not directly confirm that the protein is Sacylated per se. Further more, in most ion channels, and in fact most Sacylated proteins, the identity of the native lipid bound to a spe cific Sacylated cysteine is not known. Although palmi tate is considered to be the major lipid species involved in Sacylation, this has not been directly demonstrated in most cases, and other fatty acids, including arachi donic acid, oleate acid, and stearic acid, have also been reported to bind to cysteine via a thioester Slinkage (Linder and Deschenes, 2007;Hannoush and Sun, 2010). A major reason for this discrepancy is that mass spectrometry-based approaches to identify the native lipid specifically bound to Sacylated cysteines remain a significant challenge. This is particularly true for low abundance proteins such as mammalian ion channels, in contrast to the widespread application of mass spec trometry to directly identify native amino acids that are phosphorylated (Kordyukova et al., 2008(Kordyukova et al., , 2010Sorek and Yalovsky, 2010;McClure et al., 2012;Ji et al., 2013). As such, direct biochemical demonstration of native cys teine Sacylation is lacking in most ion channels.

S-acylation and control of the ion channel lifecycle
Ion channel physiology is determined by both the num ber of channel proteins at the cognate membrane and by their activity and/or kinetics at the membrane. Evi dence has begun to emerge that Sacylation of either pore forming or regulatory subunits of ion channels controls all of these aspects of ion channel function. Although the focus of this review is Sacylation-dependent regulation of ion channel subunits itself, Sacylation also regulates the localization or activity of many adaptor, scaffolding, and cellular signaling proteins (e.g., G protein-coupled receptors [GPCRs], AKAP18, AKAP79/150, G proteins, etc.), as well as other aspects of cell biology that affect ion channel trafficking and the activity and regulation of macromolecular ion channel complexes Linder and Deschenes, 2007;Fukata and Fukata, 2010;Greaves and Chamberlain, 2011;Shipston, 2011;Resh, 2012).
Control of ion channel cell surface expression and spatial organization in membranes. The control of ion channel trafficking, from synthesis in the ER through modifica tion in the Golgi apparatus to subsequent delivery to the appropriate cellular membrane compartment, is a and oleic acid (oleoylation) as well as myristate via amide linkages on lysine residues (Stevenson et al., 1992;Linder and Deschenes, 2007;Hannoush and Sun, 2010;Schey et al., 2010). These modifications can be discriminated from Sacylation by their insensitivity to hydroxylamine cleavage (at neutral pH) compared with the Sacylation thioester linkage. Whether N or Olinked palmitoylation or oleoylation controls ion channel function remains to be determined.
Cysteine centric (cysteine accessibility) assays: Acyl-biotin exchange (ABE) and resin-assisted capture (Acyl-RAC). The metabolic labeling approach requires treating isolated cells with lipid conjugates and thus largely precludes analysis of native Sacylation in tissues. However, several related approaches have been developed that exploit the exposure of a reactive cysteine after hydroxylamine cleavage (at neutral pH) of the cysteineacyl thioester linkage. The newly exposed cysteine thiol can then react with cysteinereactive groups (such as biotin BMCC or biotinHPDP used in the ABE approach; Drisdel et al., 2006;Draper and Smith, 2009;Wan et al., 2007) or thiopropyl sepha rose (used in AcylRAC; Forrester et al., 2011) to allow purification of Sacylated proteins that can be identified by Western blot analysis or mass spectrometry. AcylRAC has been reported to improve detection of higher mo lecular weight Sacylated proteins and thus may prove valuable for ion channel analysis. These approaches have been exploited to determine the "palmitoylome" in several species and tissues (e.g., Table 3; Wan et al., 2007Wan et al., , 2013Kang et al., 2008;Martin and Cravatt, 2009;Yang et al., 2010;Yount et al., 2010;Merrick et al., 2011;Wilson et al., 2011;Jones et al., 2012;Ren et al., 2013). For example, analysis of rat brain homogenates identi fied both previously characterized as well as novel Sac ylated ion channels (Tables 1-3). ABE and AcylRAC have recently been adapted to allow more quantita tive labeling in vivo for comparative assays (Wan et al., 2013), although it must be remembered that these approaches detect Sacylation and do not define Spal mitoylation per se. Cysteine accessibility approaches determine the net amount of preexisting Sacylated proteins; however, caution is required to eliminate false positives. In particular it is necessary to fully block all reactive cysteines before hydroxylamine cleavage; more over, the identity of the endogenously bound lipid is of course not known.
The lipid and cysteinecentric approaches are thus complementary. In conjunction with sitedirected mu tagenesis of candidate Sacylated cysteine residues in ion channel subunits, these approaches have provided substantial insight into the role and regulation of ion channel Sacylation (Tables 1-3). However, tools that are widely accessible for examination of other post translational modifications of ion channels (such as channels from the ER and the transGolgi network. De acylation at the Golgi apparatus appears to be an impor tant regulatory step . BK channel surface abundance may also be controlled by Sacylation of regu latory 4 subunits. 4 subunit Sacylation on a cysteine residue juxtaposed to the second transmembrane domain is important for the ability of the 4 subunit itself to exit the ER. Importantly, assembly of 4 subunits with specific splice variants of poreforming  subunits of the BK chan nel enhances surface expression of the channel, a mecha nism that depends on Sacylation of the 4 subunit (Chen et al., 2013). Thus, in BK channels, Sacylation of the S0S1 loop of the poreforming subunit controls global BK channel surface expression, and 4 subunit Sacylation controls surface expression of specific poreforming sub unit splice variants. Sacylation of the Kchip 2 and Kchip 3 accessory subunits also controls surface expression of volt agegated Kv4.3 channels (Takimoto et al., 2002).
Moreover, Sacylation modulates the spatial organiza tion of ion channels within membranes. Perhaps the most striking example involves aquaporin 4 (AQP4), where Sacylation of two Nterminal cysteine residues in an Nterminal splice variant (AQP4M1) inhibits assem bly of AQP4 into large orthogonal arrays (Suzuki et al., 2008;Crane and Verkman, 2009), perhaps by disrupt ing interactions within the AQP4 tetramer. Sacylation can affect the distribution of the many membrane associated proteins between cholesterolrich microdo mains (lipid rafts) and the rest of the membrane. Such clustering has also been reported for various transmem brane proteins, including the P2x purinoceptor 7 (P2X7) receptor, in which Sacylation of the C terminus pro motes clustering into lipid rafts (Gonnord et al., 2009). A similar mechanism may underlie synaptic clustering of GABA A receptors mediated by Sacylation of an intracel lular loop of the y2 subunit (Rathenberg et al., 2004). In these examples, Sacylation of the channel itself affects membrane partitioning and organization. However, re cent evidence in neurons suggests that establishment of "nano" domains of ion channel complexes in postsyn aptic membranes may also be established by local clus tering of the cognate acyltransferase itself. For example, clustering of zDHHC2 in the postsynaptic membranes of individual dendritic spines provides a mechanism for local control of Sacylation cycles of the PDZ protein adapter, PSD95, and thereby for controlling its associa tion with the plasma membrane. PSD95, in turn, can assemble with various ion channels, including NMDA receptors, and can thus dynamically regulate the local ization and clustering of ion channel complexes (Fukata et al., 2013). Indeed, an increasing number of other ion channel scaffolding proteins such as Grip1 (Thomas et al., 2012), PICK1 , Sdelphilin (Matsuda et al., 2006), and Ankyrin G (He et al., 2012) that influence ion channel trafficking, clustering, and localiza tion are now known to be Sacylated. major mechanism whereby Sacylation modulates ion channel physiology. Sacylation may influence the num ber of ion channels resident in a membrane through regulation of distinct steps in the ion channel lifecycle (Fig. 2). Indeed Sacylation has been implicated in ion channel synthesis, as well as in channel trafficking to the membrane and subsequent internalization, recy cling, and degradation. Sacylation controls the matura tion and correct assembly of ion channels early in the biosynthetic pathway. For example, Sacylation regu lates assembly of the ligand gated nicotinic acetylcho line receptor (nAChR) to ensure a functional binding site for acetylcholine (Alexander et al., 2010) as well as controlling its surface expression (Amici et al., 2012). Sacylation is also an important determinant of the maturation of both voltagegated sodium (Nav1.2) and voltagegated potassium channels (Kv1.5; Schmidt and Catterall, 1987;Zhang et al., 2007). Sacylation also con tributes to the efficient trafficking of channels from the ER to Golgi and to postGolgi transport. Three exam ples illustrate the importance and potential complexity of Sacylation in controlling ion channel trafficking: (1) Sacylation of a cysteine residue adjacent to a hy drophobic region (TM2) in a cytosolic loop of the GluA1 poreforming subunit of AMPA receptors (Fig. 4 A) pro motes retention of the channel in the Golgi (Hayashi et al., 2005). However, Sacylated Grip1b, a PDZ protein that binds to AMPA receptors, is targeted to mobile trafficking vesicles in neuronal dendrites and acceler ates local recycling of AMPA receptors to the plasma membrane (Thomas et al., 2012). In contrast, Sacyl ation of another AMPA receptor interacting protein, PICK1, is proposed to stabilize AMPA receptor internal ization .
(2) Sacylation of a cluster of cysteine residues juxta posed to the transmembrane 4 domain (Cys I site) of the NMDA receptor subunit GluN2A (Fig. 4 B) in creases surface expression of NMDA receptors by de creasing their constitutive internalization. In contrast Sacylation at Cterminal cysteine residues (Cys II site) decreases their surface expression by introducing a Golgi retention signal that decreases forward traffick ing . Even though both sites affect surface expression, only Sacylation of the TM4 juxta posed cysteine residues influences synaptic incorpora tion of NMDA receptors, which suggests that this site is an important determinant of the synaptic versus extra synaptic localization of these ion channels (Mattison et al., 2012). Together, these data highlight the impor tance of Sacylation of two distinct sites within the same ion channel as well as that of components of the ion channel multimolecular complex as determinants of channel trafficking.
(3) Sacylation of a cluster of cysteine residues in the intracellular S0S1 loop of the poreforming subunit (Figs. 4 C and 5) is required for efficient exit of BK into the plasma membrane (Lin et al., 2009). Intrigu ingly, PKC phosphorylation and Sacylation have the opposite effect on 4.1Nmediated regulation of Kainate receptor (GluK2 subunit) membrane insertion: in this, case Sacylation promotes 4.1N interaction with Kainate receptors and thereby receptor insertion, whereas PKC phosphorylation disrupts 4.1N interaction, promoting receptor internalization (Copits and Swanson, 2013). Disruption of phosphorylation by Sacylation of residues near consensus phosphorylation sites likely results from steric hindrance, as proposed for Sacylation-dependent regulation of 2 adrenergic receptor phosphorylation (Mouillac et al., 1992;Moffett et al., 1993).
Sacylation has also been reported to promote ion channel phosphorylation. For example, sitedirected mutation of a cluster of palmitoylated cysteine residues in the GluN2A subunit of NMDA receptors abrogates Fyndependent tyrosine phosphorylation at a site between TM4 and the palmitoylated cysteines . Therefore, Sacylation of GluN2A promotes tyro sine phosphorylation, resulting in reduced internali zation of the NMDA receptor . Furthermore, Sacylation of BK channels can act as a gate to switch channel regulation to different AGC fam ily kinase signaling pathways, emphasizing the complex interactions that can occur between signaling pathways (Tian et al., 2008;Zhou et al., 2012;Fig. 5). Sacylation of an alternatively spliced insert (STREX) in the large cytosolic domain of the poreforming subunit of BK channels promotes association of the STREX domain with the plasma membrane. Sacylation of the STREX insert is essential for the functional inhibition of STREX BK channels by PKAmediated phosphorylation of a serine residue immediately upstream of the Sacylated cysteines. PKA phosphorylation dissociates the STREX domain from the plasma membrane (Tian et al., 2008), preventing STREX domain Sacylation (Jeffries et al., 2012) and leading to channel inhibition. However, deacylation of the STREX domain exposes a PKC con sensus phosphorylation site downstream of the STREX domain, allowing PKC to inhibit STREX BK channels (Zhou et al., 2012). Thus, Sacylation acts as a reversible switch to specify regulation by AGC family kinases through control of the membrane association of a cyto solic domain of the channel: Sacylated STREX BK channels are inhibited by PKA but insensitive to PKC, whereas deacylated channels are inhibited by PKC but not PKA (Fig. 5). The reciprocal control of membrane associa tion of a protein domain by Sacylation and protein phos phorylation likely represents a common mechanism in other signaling proteins as revealed for phosphodies terase 10A (Charych et al., 2010).
Cysteine residues are targets for several other modi fications that regulate various ion channels, including nitrosylation, sulphydration, REDOX regulation, and for mation of disulphide bonds (Sen and Snyder, 2010).
Relatively few studies have identified effects of Sacyl ation on the intrinsic gating kinetics or pharmacology of ion channels at the plasma membrane. However, a glycinetocysteine mutant (G1079C) in the intracellular loop between domains II and III enhances the sensitivity of the voltagegated Na channel Nav1.2a to the toxins PaurTx3 and ProTxII, an effect blocked by inhibition of Sacylation. These toxins control channel activation through the voltage sensor in domain III. In addition, de acylation of another (wildtype) cysteine residue (C1182) in the II-III loop produces a hyperpolarizing shift in both activation and steadystate inactivation as well as slowing the recovery from fast inactivation and increasing sensitivity to PaurTx3 (Bosmans et al., 2011). Effects of Sacylation on gating kinetics have also been reported in other channels. For example, in the voltagesensitive potassium channel Kv1.1, Sacylation of the intracellular linker between transmembrane domains 2 and 3 increases the intrinsic voltage sensitivity of the channel (Gubitosi Klug et al., 2005). Sacylation of the  and  subunits of epithelial sodium channels (ENaC) also affects channel gating (Mueller et al., 2010;Mukherjee et al., 2014), and the Sacylated regulatory 2a subunit of Ntype calcium channels controls voltagedependent inactivation (Qin et al., 1998;Hurley et al., 2000).
Sacylation is also an important determinant of re trieving ion channels from the plasma membrane for recycling or degradation. Sacylation of a single cysteine residue juxtaposed to the transmembrane TM4 domain of GluA1 and GluA2 subunits of AMPA receptors con trols agonistinduced ion channel internalization. These residues are distinct from those controlling Golgi reten tion of AMPA receptors (Fig. 4 A), which emphasizes the finding that the location and context of the Sacylated cysteines, even in the same protein, is central for their effects on physiological function (Hayashi et al., 2005;Lin et al., 2009;Yang et al., 2009). The stability of many proteins is also regulated by Sacylation; Sacylation of a single cysteine residue in Kv1.5 promotes both its inter nalization and its degradation (Zhang et al., 2007;Jindal et al., 2008). Thus, in different ion channels, Sacylation can have opposite effects on insertion, membrane stabil ity, and retrieval.
S-acylation and posttranslational cross-talk control channel trafficking and activity. An emerging concept is that Sacylation is an important determinant of ion channel regulation by other PTMs. Indeed, nearly 20 years ago it was reported that PKCdependent phosphorylation of the GluK2 (GluR6) subunit of Kainate receptors was attenuated in channels Sacylated at cysteine residues near the PKC consensus site (Pickering et al., 1995). Sacylation of GluA1 subunits of AMPA receptors also blocks PKC phosphorylation of GluA1 and subsequently prevents its binding to the cytoskeletal adapter protein 4.1N, ultimately disrupting AMPA receptor insertion this is determined by interaction with an Sacylated R7 binding protein (R7BP) that acts as an allosteric activa tor. Thus, the R7RGS complex, recruited to the plasma membrane by Sacylated R7BP, promotes Gi/o deacti vation to facilitate GIRK channel closure. Conversely, deacylation of R7BP removes the R7GS complex from the plasma membrane, slowing Gi/o deactivation and consequent channel closure (Jia et al., 2014). Clearly, as Sacylation can also control an array of GPCRs, enzymes, and signaling and adapter proteins that indirectly con trol ion channel function Linder and Deschenes, 2007;Fukata and Fukata, 2010;Greaves and Chamberlain, 2011;Shipston, 2011;Resh, 2012), understanding how Sacylation dynamically con trols other components of ion channel multimolecular signaling complexes will be an essential future goal.

Summary and perspectives
With an everexpanding "catalog" of Sacylated ion channel poreforming and regulatory subunits (50 to date), together with an array of Sacylated scaffolding and signaling proteins, the importance and ubiquity of this reversible covalent lipid modification in controlling the lifecycle and physiological function and regulation of ion channels is unquestionable. This has been par alleled by a major resurgence in the wider Sacylation field, a consequence in large part of the discovery of Sacylating and deacylating enzymes together with a growing arsenal of genetic, proteomic, imaging, and pharmacological tools to assay and interrogate Sacyla tion function.
As for most other posttranslational modifications of ion channels, including phosphorylation, major future goals for the field include: (1) Understanding mechanistically how covalent ad dition of a fatty acid can control such a diverse array of ion channel protein properties and functions, and how this is spatiotemporally regulated.
(2) Elucidating the physiological relevance of this posttranslational modification from the level of single ion channels to the functional role of the channel in the whole organism in health and disease.
Elucidation of these issues has fundamental implica tions far beyond ion channel physiology.
To address these goals several major challenges and questions must be addressed, including: (1) It is largely assumed that Sacylation of trans membrane proteins results in an additional "membrane anchor" to target domains to the membrane interface. However, understanding the mechanisms, forces, and impact of Sacylation on the orientation of transmem brane helices and the architecture and structure of dis ordered domains in cytosolic loops and linkers, while remaining a considerable technical challenge, should provide major insight into mechanisms controlling chan nel trafficking, activity, and regulation.
Evidence is beginning to emerge that Sacylation may mutually compete with these mechanisms, providing a dynamic network to control cysteine reactivity. For ex ample, the ion channel scaffolding PDZ domain protein PSD95 is Sacylated at two Nterminal cysteine residues (C3 and C5) that are required for membrane target ing and clustering of PSD95 (ElHusseini et al., 2002). nNOS also interacts with PSD95, and stimulation of ni tric oxide production results in nitrosylation of these cysteines, preventing their Sacylation and thereby de creasing PSD95 clusters at postsynaptic sites (Ho et al., 2011). A recent remarkable example of the potential for such crosstalk in ion channel subunits is the identifica tion of the Sacyl ation of 18 different cysteine residues in the large cytosolic N terminus of RyR1 in skeletal muscle. Of these 18 Sacylated cysteines, six have pre viously been identified as targets for Soxidation, and a further cysteine residue was also subject to Snitrosyl ation (Chaube et al., 2014) Although the functional rel evance of this potential crosstalk in RyR1 has yet to be defined, interaction between oxidation and Sacylation of the same cysteine residue is physiologically relevant in other proteins. For example, oxidation of the signal ing protein HRas at two cysteine residues C181/184 prevents Sacyl ation of these residues, resulting in a loss of plasma membrane localization of this peripheral membrane signaling protein (Burgoyne et al., 2012). In triguingly, a conserved cysteine residue in nAChR 3 subunits, which has been shown to be Sacylated (C273) in the nAChR 4 subunit, has been implicated in use dependent inactivation of nAChRs by reactive oxygen species (Amici et al., 2012). Determining whether these mutually competitive cysteine modifications represent an important mechanism for regulation of a range of ion channels is an exciting challenge for the future.
Sacylation is also an important determinant of ion channel regulation by heterotrimeric G proteins. This can involve Sacylation of either G protein targets or of regulators of G proteins. In an example of the for mer, the palmitoylated N terminus of the regulatory 2a subunit splice variant acts as a steric inhibitor of an arachidonic acid binding domain to stimulate Ntype calcium channels (Chien et al., 1996;Heneghan et al., 2009;MitraGanguli et al., 2009). When the regulatory  subunits are not Sacylated, however, Gqmediated sig naling, via arachidonic acid, inhibits calcium channel activity. Closure of G protein regulated inward rectify ing potassium (GIRK) channels in neurons after Gi/o deactivation provides an example of the latter (Jia et al., 2014). Signaling by members of the Gi/o family of the G subunit of heterotrimeric G proteins is terminated by members of the regulator of G protein signaling 7 (R7 RGS) family of GTPaseactivating proteins, which accelerate GTP hydrolysis to speed Gi/o deactivation. Membrane localization of regulator of G protein signal ing 7 (R7RGS) is required for its regulation of Gi/o, and paradigm shift in our understanding of both ion channel and Sacylation physiology, and promises to reveal novel therapeutic strategies for a diverse array of disorders.
Work in the author's laboratory is generously supported by the Wellcome Trust (grant No. WT082407MA), Medical Research Council, and British Heart Foundation.
The author declares no competing financial interests.
Elizabeth M. Adler served as editor.
Submitted: 30 January 2014 Accepted: 18 April 2014 (2) Although Sacylation is widely accepted to be re versible, its spatiotemporal regulation of most ion chan nels is unknown. Mechanistic insight into zDHHC and acylthioesterase substrate specificity, native subcellular localization, and assembly with ion channel signaling complexes will allow us to dissect and understand how Sacylation of ion channels is controlled. Importantly, this should allow us to take both "channelcentric" (e.g., sitedirected mutagenesis of Sacylated cysteines) as well as "Sacylation centric" (e.g., knockout of specific zDHHC activity) approaches to understand how multisite Sacyl ation on the same ion channel subunit can control distinct functions as well as physiological regulation of trafficking and function at the plasma membrane.
(3) The functional role of Sacylation cannot be viewed in isolation from other posttranslational modifications. The crosstalk between Sacylation and adjacent phosphor ylation sites as well as other cysteine modifications high lights the importance of understanding the interactions between signaling pathways. Insight into the rules, mecha nisms, and crosstalk of Sacylation with these modifica tions has broad implications for cellular signaling.
(4) Although it is clear that disruption of Sacylation homeostasis itself has substantial effects on normal phys iology, and we are beginning to understand some of the cellular functions of ion channel Sacylation, we know very little about the functional impact of disrupted ion channel Sacylation at the systems and organismal level. Understanding how this may be dynamically regulated during a lifespan is critical to understanding the role of Sacylation in health and disease.
To address these issues, development of improved tools to assay and investigate Sacylation from the single protein to organism is required. For example, tools to allow the realtime analysis of Sacylation status of ion channels in cells and tissues will provide fundamental insights into its dynamics and role in ion channel traf ficking and membrane localization. Improved pro teomic tools will allow direct assay of fatty acids bound to cysteine residues via thioester linkages. Development of new tools and models are essential if we are to under stand the physiological relevance of ionic channel Sac yl ation at the systems level. These include: specific inhibitors of zDHHCs and thioesterases, conditional knockouts to spatiotemporally control zDHHC expres sion, and transgenics expressing catalytically inactive zDHHCs and models expressing Sacylation-null ion channel subunits. Furthermore, our understanding of how Sacylation may be dynamically controlled during normal ageing in response to homeostatic challenge and disruption in disease states remains rudimentary. Whether we will start to uncover channel "Sacylation opathies" resulting from dysregulation of ion channel Sacylation, analogous to channel phosphorylopathies, remains to be explored. Addressing these issues, to gether with development of new tools, will provide a