Lipid Agonism, The PIP2 Paradigm of Ligand-Gated Ion Channels (original) (raw)

. Author manuscript; available in PMC: 2016 May 1.

Published in final edited form as: Biochim Biophys Acta. 2015 Jan 26;1851(5):620–628. doi: 10.1016/j.bbalip.2015.01.011

Abstract

The past decade, membrane signaling lipids emerged as major regulators of ion channel function. However, the molecular nature of lipid binding to ion channels remained poorly described due to a lack of structural information and assays to quantify and measure lipid binding in a membrane. How does a lipid-ligand bind to a membrane protein in the plasma membrane and what does it mean for a lipid to activate or regulate an ion channel? How does lipid-binding compare to activation by soluble neurotransmitter? And how does the cell control lipid agonism? This review focuses on lipids and their interactions with membrane proteins, in particular ion channels. I discuss the intersection of membrane lipid biology and ion channel biophysics. A picture emerges of membrane lipids as bona fide agonists of ligand-gated ion channels. These freely diffusing signals reside in the plasma membrane, bind to the transmembrane domain of protein, and cause a conformational change that allosterically gates an ion channel. The system employs a catalog of diverse signaling lipids ultimately controlled by lipid enzymes and raft localization. I draw upon pharmacology, recent protein structure, and electrophysiological data to understand lipid regulation and define inward rectifying potassium channels (Kir) as a new class of PIP2 lipid-gated ion channels.

Keywords: Lipid gated, Ion channel, PIP2, Signaling lipid, G-protein, Lipid raft, Lipidomics

1. Introduction

Signaling lipids are important regulators of ion channels and exert a central role in tissue function including functional heartbeat, neuronal signaling, kidney dialysis, sight, smell, pain, and touch [1–5]. In the past, most biochemist and ion channel experts viewed lipids as unwieldy, hydrophobic molecules physically supporting ion channels in a cell membrane or liposomes but not as ligands. Recent past models of lipid signaling to ion channels suggested that the formation of anionic lipids caused a change in the plasma membrane surface charge. Little was known about how lipids engaged and disengaged the channel or how the contact of a lipid with protein might affect the conformation of ion channels in the membrane. A lack of binding constants for lipids and ion channels challenged our ability to think about lipids as ligands. Aspects of this problem remain an important hurdle.

In 1998 Hilgemann and colleagues eloquently showed that a signaling lipid could directly activate an ion channel [6]. The lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), a minor constituent of the plasma membrane, was required and sufficient for the activation of a potassium channel [6]. Despite more than a decade of experimentation, the nature of PIP2 binding remained clouded by an inability to accurately measure its concentration in the membrane and directly detect binding to protein. Simple terminology such as lipid concentration and affinity are difficult to define for insoluble molecules in an aqueous environment [7]. Absent a well-characterized ligand protein interaction; initial non-specific theories of surface charge and membrane curvature dominated [8,9] but struggled to account for the specificity of signaling lipids in many systems. Recently a more accurate model emerges that includes structural and pharmacological evidence that lipids bind to and activate ion channels analogous to classic ligand-like agonist properties [10,11].

Herein a model of lipid agonism is built on PIP2 and inward rectifying potassium (Kir) channels. Aspects of many other classes of channels and signaling lipids appear to function in a similar way; select examples are included throughout this review. The intent of this review is to facilitate an understanding at the interface of ion channel activation and membrane lipid biology, although neither field is reviewed in a comprehensive way.

2. The signaling lipid PIP2 is an agonist that gates ion channels

PIP2, arguably the best-studied signaling lipid, is comprised of an inositol head group (the named feature) a phosphoglycerol backbone, and two acyl chains (Fig. 1A). PIP2 bears four negative charges and is a permanent and minor component (<1%) of the Eukaryotic plasma membrane inner leaflet [12,13].

Fig. 1.

Fig. 1

PIP2 lipid regulation of ion channels. A, The chemical structure of plasma membrane PIP2 is shown with an arachidonyl acyl chain (green) and inositol phosphates at the 4′ and 5′ position (red). B, a cartoon representation of a PIP2 lipid gated ion channel. PIP2 is shown bound to a lipid binding site in the transmembrane domain of an ion channel. C, List of ion channels with lipid gating properties. Kir2.2 and 3.2 are the most clearly “lipid-gated”. A second group appears to be dual regulated, or “PIP2 modulated”. PIP2 modulates channel gating, but gating also requires either voltage or a second ligand. A third group of channels behave similar to Kir but await definitive proof of lipid gating vs. PIP2 modulation (?). The list of channels is exemplary and not comprehensive.

2.2 PIP2 ion channel physiology

PIP2 signaling dictates the activatable state of a plethora of ion channels [2,14,15] (Fig. 1) with broad reaching cellular function. The first indication that a channel is PIP2 dependent usually arises when a channel, excised from the plasma membrane (e.g. inside out patch), steadily decreases in conductance until the channel inactivates, this is known as “rundown” [2,16]. The excised patch lacks the cytosolic factors to maintain sufficient PIP2 levels in the membrane to support ion channel function; hence the channels in the patch close. Adding ATP and Mg was shown to delay rundown [16]. Presumably, PIP2 synthesizing enzymes are excised in the patch with the channels and that these enzyme utilize the ATP to replenish PIP2 [2,16]. Adding back a soluble PIP2 analog dioctanoyl PIP2 (C8PIP2) rescues activity [2,15] of many ion channel types [17–20]. In a second method, PIP2 scavengers (e.g. polyamines or PIP2 antibodies) are used to deplete or mask PIP2 availability [21–23]. Polyamines are positively charged polymers that bind via avidity to the multiple negative charges of PIP2. More complete descriptions of PIP2 dependent ion channels and PIP2 cellular function are reviewed by Suh and Hille [2,11], Xie [5], and McLaughlin [9]. Recently, a voltage sensitive phosphatase (Ci-VSP) was shown to provide direct control over PIP2 signaling in the membrane [24–26]. When Ci-VSP is co transfected with Kir [24–26], Kv7.1 [27], Cav2 [28,29], and TRP [30,31], channels are voltage dependent consistent with Ci-VSP regulation of PIP2. This method provides better control of PIP2; however, indirect effects of PIP2 remain a possibility.

In order to directly show PIP2 modulation, an ion channel can be purified and reconstituted (reinserted) into lipid vesicles with a known lipid composition. A lack of purified ion channels limited this technique, but recent advancements in membrane protein expression and purification [32,33] has overcome this problem for select channel types [34–38]. The nAChR was among the first channels to show direct dependence on a lipid for activation, phosphatidic acid (PA) [39]. Recently PIP2 dependent channels were reconstituted into lipid vesicles and shown to respond directly to PIP2 modulation, this includes GIRK [40,41], TRPV1 [42], TRPM8 [43], and Kir2.1-2 [44] channels.

2.3 PIP2 ion channel structure

Despite robust channel modulation by indirect methods, absent a crystal structure, an understanding of the molecular action of PIP2 and the precise binding site remained speculative. In 2011 an X-ray crystal structure complex of Kir2.2 with PIP2 revealed a PIP2 binding site in the channel’s transmembrane domain [10] (Fig. 2). The glycerol backbone and 1′ phosphate of PIP2 capped the first transmembrane spanning helix (TM1) of Kir. Intimate coordination of the 5′ inositol phosphate in the distal end of the second transmembrane spanning helix (TM2) accounted for PIP2 specificity. And a conformational change appeared to initiate or open the ion conduction pathway. Basic residues on a linker between the transmembrane domain and cytoplasmic domain directly contacted PIP2, but distal basic residues proposed in the CTD [45] did not, rather they were buried and stabilized proper folding of the cytoplasmic domain structure [10]. Prior to the Kir2.2/PIP2 complex, structures of PIP2/protein complexes were limited to soluble membrane localization domains, which lack a transmembrane domain and share few if any functional similarities with ion channels. A lack of appropriate structural examples and an understanding of how lipids and proteins interact in the plasma membrane hindered a complete mechanistic interpretation of PIP2 data. Furthermore, early studies on the C-terminus of Kir included residues that turned out to be in the TMD of Kir and key to binding the 5′ inositol phosphate [6] (Fig. 2). Only with recent structural data has a model emerged where lipids bind to specific sites in the transmembrane domain of ion channels [10,46–49].

Fig. 2.

Fig. 2

Conserved PIP2 binding site in Kir2.2. PIP2 binds the transmembrane domain (TMD) of Kir and causes a conformational change that allosterically gates the channel. A, The PIP2 binding site is specific for inositol 5′ phosphate. B, A sequence alignment of all Kir family members reveals a highly structured PIP2 binding site comprised of basic residues. Amino acid residues that directly contact PIP2 are shown in bold type. Only two residues (brown type) at the conserved site lack a positive charge. Residues originating from the TMD and a linker (LNK) are shaded green and grey respectively. ^ indicates residues that strongly coordinate the lipid backbone phosphate, * indicates the residues that strongly (red) and weakly (grey) bind the PIP2 5′ phosphate. PIP2 atoms are colored yellow, carbon, orange phosphate; red oxygen. Amino acid side chains with carbons colored green are located on transmembrane outer helix 1 (TM1) or inner helix 2 (TM2). Lysines colored grey are located on the start of a linker helix (LNK) or “tether helix” connecting the transmembrane domain (TMD) and the cytoplasmic domain (CTD). Residue numbering is according to Kir2.2.

2.4 Lipid-gating theory

Taken together these finding suggest a ligand-gating theory of PIP2 activation. In biochemistry, the term ligand refers to the reversible, specific, and dose dependent binding of a substance to a protein to form a complex. Ligands include small molecule drugs, hormones, peptides, and metabolites. Normally ligands stabilize at least two states, one bound and one unbound [50,51].

The binding of PIP2 to Kir has many features of a ligand. First, PIP2 is in low abundance [9,12]. This requires that PIP2 bind with high affinity to its targets to exert an effect. Second, PIP2 binds reversibly to ion channels in a dose dependent manner [20,23]. Third, PIP2 binds with specificity; for example, PI(4,5)P2 activates Kir2.1 and PI(3,4)P2 inhibits the same channel [52]. This specificity is striking since the two lipids are chemical isomers and only differ in the position of the 5′ phosphate. Another anionic lipid, oleoyl-CoA, competitively and reversibly inhibits all Kir’s [52] except Katp, which is specifically activated by oleoyl-CoA [53,54]. Fourth, like neurotransmitter, PIP2 is a dynamically regulated molecule [55,56]; a signaling cascade can rapidly change the concentration of PIP2 to cause the channels to open or close [57–59]. And lastly, PIP2 channel affinity determines channel function [60]. Mutations that allosterically decrease the affinity of PIP2 cause disease (e.g., Andersen-Tawil syndrome) [45,61].

The ligand-like characteristics of PIP2 binding to the entire family of inward rectifiers warrant classification of these channels as ligand-gated. The unique properties of lipids logically give rise to a lipid subclass suggested here “lipid-gated” ion channels.

3. The evolving view of PIP2

3.1 Membrane surface charge theory

PIP2 was first speculated to induce ion channel activation by non-specific avidity of negatively charged phospholipid binding to clusters of basic amino acids in the C-terminus of channels [2,5,8]. Anionic lipids were thought to accumulate on the inner leaflet and non-specifically attract positively charged residues on the surface of Kir’s cytoplasmic domain (CTD). The rational for the theory is sound and was based on data from Katp (Kir7.x) [21,62–64] and proteins like MARCKS [2,8]. However, in light of the PIP2/Kir complexes, the previous role of electrostatic theory appears inadequate for Kir. The glycerol backbone of PIP2 bound tightly to the transmembrane domain (TMD), and the inositol phosphates interacted with residues in or proximal to the TMD, not the CTD. The original influential lack of Katp’s specificity is an anomaly among Kir’s and appears to be an adaptation that allowed regulation by oleoyl-CoA [20] and not a mechanistic requirement as speculated. If non-specific anionic interactions regulate Kir, the site of anion lipid binding are likely distal to the canonical PIP2 site [65] or act synergistically with PIP2 [44,66] by binding to one of the 4 canonical sites. The notion that the cytoplasmic domain is the binding site for PIP2 and that PIP2 localizes the CTD similar to a PH domain appears to be incorrect. The Kir2.2 CTD did move toward the membrane and may reflect an evolutionary origin; but the primary mechanism appears to be an allosteric conformational change, not non-specific electrostatic attractions of the CTD to the membrane surface. The key PIP2 binding interactions were confirmed in a complex of PIP2 with GIRK2 [48] suggesting a common mechanism in related Kirs (Fig. 2B).

Voltage activated ion channels better exemplify non-specific electrostatic interaction. A well studied domain called the “voltage sensor domain” (VSD) senses and responds to changes in surface charge [33,47,67,68]. Conserved basic residues in the VSD electrostatically move towards the charge causing a conformational change that gates the channel. The charge is non-specific and can be applied by external current or by changing the charge of lipids in the plasma membrane. The latter was shown in recent bilayers studies where Kv responded symmetrically and non-specifically to anionic lipids [69]. The same study showed a distinct phosphatidic acid site in the cytoplasmic leaflet that specifically and dramatically affected Kv gating [69]. This suggests both ligand and electrostatic modes can operate in the same channel, however the structural determinants of the two are likely distinct. A similar arrangement exists in Cav2, which has a voltage sensor and a putative PIP2 specific binding site [11,70].

Few other channels currently have sufficient molecular description to definitively discriminate the mechanism of action seen in Kir and Kv. Many tetrameric channels exhibit a C-terminal charged cluster and varying degrees of specificity reminiscent of Kir, including TRP [19,42,71–75], and P2X4 [76,77] (see table 1). Typically, these charges immediately follow or are located in the last transmembrane domain. Many other channels respond to PIP2 in ways that parallel Kir responses, including Cav [70], NMDA [78], Kv [27], P2X1-3 [79] channels (see also Fig. 1C), but it is unknown if the interactions are direct with the TMD or indirect through membrane charge or other proteins. Since numerous soluble domains use polybasic clusters to target to the plasma membrane [80], some yet undefined cytoplasmic domains could utilize a membrane surface charge as previously speculated [2,8]. Future structural studies will continue to reveal the details and breadth of electrostatic theory.

Table 1.

Insositolphosphate ion channel specificity

Channel PIP2 effect Selectivity over Comments Ref
TRPM8 Activation* PI(3,4)P2 and PIP3 5′ activates, 3′ inhibits [19,43]
TRPV1 Mixed PI(4)P and PIP3 Likely acyl chain dependence [42,71]
TRPM4 Activation PI(4)P and PI(5)P Modest selectivity over PIP3 [75]
P2X4 Activation PIP3 Modest selectivity over PIP3 [77]
TRPML Inhibition PI(3,5)P2 (activation) Direct competition of (3,5) with PI(4,5)P2 [72,73]
Kir2.1,1.1 Activation* PI(3,4)P2 (inhibition) Direct competition of (3,4) with PI(4,5)P2 [20,52]
Kir3 (Girk2/4) Activation* PI(4)P Gbg increases PI(4,5)P2 binding [20]

3.2. Cofactor theory

Lipids are sometimes viewed as co-factors. Before discussing PIP2 as a cofactor I must first define a cofactor and distinguish it from a ligand. The term cofactor stems from enzymology and generally refers to a permanent organic compound or metal that is required for the enzyme to function. A cofactor normally derives its function by remaining bound to a protein. In contrast, a ligand derives its function by binding and dissociating from its partner protein. Lipids have always existed in cells and it is reasonable to assume that some lipids may bind as cofactors. A crystal structure of Kv in a lipid like environment revealed phospholipid binding sites near the voltage sensor and some of these appear to be lipid cofactors [47]. In other words they facilitate the proper organization of the channel but at present they do not appear to initiate a change in the channel state by dynamic regulation of the lipid.

In a speculative role, PIP2 was proposed to act as a ‘coincidence detector’ in order to facilitate transport of an inactive channel [2,15,81,82]. A nascent channel in the endoplasmic reticule (ER), where PIP2 is scarce, remains inactive until it arrives, at the plasma membrane where an abundance of PIP2 constitutively activates the ion channel. This fits well a definition of cofactor in the resting state. Directly demonstrating the physiological contribution remains a challenge since PIP2 is dynamically regulated [2]. For example PLC hydrolysis of PIP2 in the plasma membrane inhibits Kir [58,59], a function also consistent with ligand-like properties.

In another speculative role, PIP2 might function as a cofactor in sensing protons. The pka’s of inositol phosphates are around 6.5 and 6.9, an optimal range for sensing physiological changes in proton concentration [83]. The lipid could remain bound and simply supply the metal phosphate as a proton sensing cofactor. Ions interacting with lipids were recently shown to regulate a receptor [84]. Acid sensing ion channels (ASIC) are likely candidates for such a mechanism since they bind PIP2 and sense protons. Alternatively, PIP2 may serve as a proton sensitive ligand. An atomic structure is known for ASIC [38] but the role of PIP2 in channel activation requires further investigation.

Perhaps one reason for a slow adaptation of a “lipid-gating” model for PIP2 is the fact that the prototypical PIP2 gated channel Kir is active during the resting state of excitable cells. These channels are often considered “constitutively active” leak channels. While it is true they allow potassium out of the cell during the resting state, acetylcholine stimulation of M1 muscarinic receptor inactivates Kir [58,59]. An early study on high affinity Kir2.1 in oocytes showed resistant to ACh inactivation [60], but later studies in mammalian cells demonstrated robust and complete inhibition of Kir2.1 through activation of M1 receptor [59]. Thus neurotransmitter induced closure of Kir potassium channels is presumably synergistic with the opening of calcium, sodium and voltage-gated channels, and should result in a stronger action potential or sustained excitability.

4. Cellular regulation of PIP2 agonism

The agonist properties of lipids broaden the cell-signaling role of PIP2 regulation. Similar to neurotransmitter, the release, degradation, and localization of PIP2 must govern ion channel function.

4.1 Lipid mediated localization of PIP2 in the plasma membrane

Phosphoinositides distributes heterogeneously in the plasma membrane [85–87]. Hydrophobicity causes lipids to partition (see Fig. 5). Saturated lipid chains partition into cholesterol rich lipid rafts, often referred to as detergent resistant membranes (DRMs). Lipids with unsaturation partition into the liquid disordered phase (Ld). Mass spec of resting cells indicate that PIP2 is comprised of a polyunsaturated fatty acyl chain [88–90] and localizes in the Ld region of the membrane [87]. Quantitative studies of PIP2 suggest close to 85% of PIP2 is polyunsaturated and 70% comprised of an arachidonyl acyl chain [90]. In contrast, PIP3 is primarily comprised of saturated or monounsaturated lipid acyl chains [89]. Strikingly, arachidonyl PIP3 was not detected in quiescent cells [89]. Based on standard lipid partitioning, the saturated PIP3 is likely located in cholesterol rafts. In agreement with this arrangement, PI3 Kinase (the enzyme that generates PIP3 from PIP2) localizes to lipid rafts [91]. Taken together, these data indicate an acyl chain based localization of PIPs in the plasma membrane. Figure 4 shows a hypothetical layout of the quiescent cell based on available, but limited, mass spec, super resolution imaging, and localization studies [87–90].

Fig. 5.

Fig. 5

PIP2 transient signaling. A, In the proposed model, PIP2 dissociates from Kir and diffuses laterally in the plasma membrane. G proteins activate lipid-hydrolyzing enzymes that deplete PIP2 from the plasma membrane or laterally redistribute PIP2 into distinct lipid micro domains (e.g. lipid rafts). Dynamic PIP2 signaling gives rise to a transient inactivation of Kir that contributes to an action potential. B, PIP2 degradation products are taken up by endocytosis and PIP2 resynthesis returns the cell to a resting state.

Fig. 4.

Fig. 4

Phosphoinositide (PI) partitioning in the plasma membrane. In the absence of a stimulus, arachidonyl-PIP2 (green) localizes in the disordered region of the plasma membrane and sometimes in concentrated lipid micro domains (dark green) apart from cholesterol-rich lipid-rafts (red). Inositol lipids are distributed according to their acyl chains; hence, saturated PIP2 enters lipid rafts where PI3 kinase generates PIP3. A saturated lysoPIP2 may also associates with raft like domains. Key signaling enzymes (see colored boxes) appear localized in lipid micro domains where they are optimally positioned to remodel the PIP acyl chains and head groups during signaling. Lipid degradation products are found in endocytic vesicles, which suggest a lipid-recycling event analogous to recycling of some soluble neurotransmitters. Grey diamond represents PI3 kinase.

4.2 GPCR signaling through lipases

Famously, Gq coupled GPCRs (guanine nucleotide coupled receptors) hydrolyze PIP2 through phospholipase C (PLC) activation. G protein mediated PIP2 hydrolysis was known more than 30 years ago [92]. However, most cell biologist viewed (and many still do) PIP2 as little more than a substrate for second messenger signaling [93]. This view is inadequate for Kir channels; PIP2 must also be viewed as an ion channel activator [3,6] or agonist. Hence, hydrolysis of PIP2 by M1 muscarinic receptors should be viewed as a direct regulatory mechanism to deplete agonist. PIP2 hydrolysis inactivates both high and low affinity Kir channels [58,59]. Downstream modulation of Kir by phosphatases and kinases appear secondary to this direct PIP2 regulation [6,94], a rational also supported by the central and highly conserved role of PIP2 in channel activation as described above (2.5). PLC regulation of Cav [70], Kir [95], HCN [96], Kv7 [27], K2P [97], and TRP [98,99] channels (among others) is well-documented.

In addition to PLC, GPCR signaling activates phospholipase D [100] (PLD). PLD produces PA and free choline. PA has emerged as an important signaling lipid [101]. PA and PIP2 appear to synergistically activate Kir [44] and K2P [102] channels, in contrast the nAChR [39] and some Kv [69] respond specifically to PA and not PIP2. A third important class of lipases phospholipase A2 (PLA2) also exhibits GPCR regulation [103]. PLA2 hydrolyzes arachidonyl-lipids creating lysophospholipids and arachidonic acid. Downstream and second messenger signaling are well studied for PLA2 and PLC and include the arachidonic cascade and IP3 second messenger signaling respectively. In comparison the upstream role of the intact bioactive arachidonyl-phospholipids and PIP2 is much less understood. Nonetheless, the added role of PIP2 in directly gating ion channels solidifies a direct rout for GPCR regulation of ion channels independent of downstream kinases and calcium signaling [6,94].

Several ion channels bind G-proteins directly, this role is widely accepted for the G-protein regulated inward rectifiers (GIRK/Kir3.x) and N-type calcium channels (Cav2) [104]. A trimeric complex of GIRK with Gβγ (a G-protein) and PIP2 revealed the GIRK/Gβγ interface [40]. And biochemical studies suggest that Gβγ is important for increasing binding of PIP2 to GIRK [41]. The precise mechanism by which Gβγ enhances PIP2 activation needs further clarification.

4.3 Protein mediated localization of lipid modifying enzymes

Lipases localize with ion channels to increase the speed and specificity of PIP2 channel gating [98,105]. For example rhodopsin activated PLC hydrolyzes PIP2 opening TRPL channels. Colocalization of PLC with TRPL [98] allows for a fast 20ms response time [106]. And TRPM7 directly binds PLC to locally affect channel activation [94]. PLC functionally colocalized with NMDA receptors [78] and the IP3 receptor co-localizes with PLC to regulate calcium release [107]. PLD lipases directly localize to ion channels, including TRPM8 [108] and TREK-1 [109]. There are many subtypes of lipases; their diverse regulation and specific localization satisfies cells with the needed diversity for signaling.

4.4 Transient PIP2 signaling

The partitioning of PIPs and their modifying enzymes appears primed to deliver dynamic cell signaling. During a signaling event, G-proteins control PIP kinases, lipases, and phosphatases, to degrade PIP2 signaling. This signaling generates lipid degradation products (Fig. 5B). For example, it was shown PLC activation generates arachidonyl-diacyl-glycerol [90]. And PLA2 activation removes the arachidonyl sn2 acyl chain generating lysoPIP2 [110]. In order to return to a resting state, degradation products need to be removed from the membrane and PIP2 resynthesized.

Endocytosis recycles lipid micro domains and lipid rafts after signaling [111]. The late endosome and ER feed back into PIP2 signaling. This postsynaptic lipid reuptake would then reset the membrane for another signaling event analogous to presynaptic neurotransmitter reuptake. Further studies are needed to understand the temporal and spatial regulation of PIP2 in vivo in particular during a signaling event. However, signaling lipids are known to control the ion channel desensitization [19], voltage dependence [75], and recovery from inactivation [94], and these events correlate with ion channel rundown. Lipid regulated desensitization may prove to be a central function of many channel types. Much more data is needed to build a complete picture.

4.5 Other mechanistic considerations

Lipids localize topically by leaflets generating a lipid signal. For example phosphatidylserine (PS) is found on the inner leaflet of the plasma membrane. Enzymes known as flippases and floppases move lipids between leaflets [112]. PS signals by flipping outside the cell [113]. PS is negatively charged and movement outside the cell has the ability to change the membrane surface charge from negative inside to negative outside. Recently, asymmetric changes to the charge of lipids in a bilayer dramatically shifted the voltage midpoint potential of a Kv channel[69]. Hence lipids may “flip” as a rapid mechanism to impose a lipid induced change on the cell membrane potential, a mechanism that would have likely preceded a synapse.

In a separate mechanism, lipid acyl transferases (LAT) could signal to ion channels by changing the unsaturation of a lipid acyl chain. LAT enzymes add acyl chains to lipids or move acyl chains between existing lipids [114]. If a LAT enzyme swaps an arachidonyl acyl chain with a saturated one, the signaling lipid would most likely translocate to a lipid raft (Fig. 5). This may simply sequester the signal away from the ion channel by moving the lipid into or out of a lipid micro domain. Alternatively, the translocation could make the lipid available to other modifying enzymes that would then deplete the signal from the membrane.

Or, lipid acyl chains may directly contribute to gating of an ion channel. The acyl chains contain chemical diversity and putative specificity could determine the affinity of the lipid for the channel or cause a specific conformational change that gates the channel. Hydrophobic sites for lipid acyl chains affect PIP2 activation of Cav2.2 [11].

The four identical binding sites in Kir are positioned for PIP2 cooperatively and allosteric competition. Tetrameric channels engineered to have only one binding pocket indicated that one PIP2 molecule is sufficient to activate the channel [66]. In wild type channels with four binding sites, PIP2 in combination with PA, PG, or PS, dramatically increased channel conductance. However, absent PIP2, these lipids failed to activate Kir [44]. A structure of Kir with PA bound showed PA binding to the canonical PIP2 site [10]; a site also compatible with PG and PS. In biochemical studies, oleoyl-CoA, an endogenous inhibitor, also competes directly with PIP2 [52]. Taken together, these studies suggest that in Kir the lipid binding site is always occupied, and Kir integrates the sum total of the lipid environment in a cooperative way. At least one site must be occupied by PIP2; the remaining three canonical sites appear to be available to exert cooperative activation or inhibition through a rigid conformational change [10] in the CTD. Thus additional PIP2 binding events are poised to activate Kir with increasing affinity consistent with electrophysiology recordings [66].

Lastly, the relative abundance of diet-derived fatty acids may affect the levels of PIP2 signaling in the plasma membrane. Cells appear to incorporate the relative amounts of saturated and unsaturated fats into their cell membranes (phospholipids) [115]. It is tempting to speculate that diets with excess saturated fat would lead to saturated PIP2 signaling, which most likely favors PIP3 signaling. Diets with large amounts of polyunsaturated fats (PUFAs) would lead to more arachidonyl-PIP2 and more PIP2 signaling. This may account for the positive affect of dietary PUFAs on heart arrhythmias and insulin resistance since PIP2 channels (including Kir) are central to both these diseases. Consistent, with this model, loss of PIP2 channel activation is associated with the disease states [61]. Similar speculation could be made of chronic pain and perhaps some cancers. Understanding PIP2 acylation may shed light on these important medical problems.

5. The future of lipid Ion channel interactions

5.1 Pharmacology of lipids

Better methods are needed for assaying lipid interactions with ion channels. Most studies rely on crude pharmacological shifts of PIP2 concentrations in biological membranes; this is inadequate. Varying the concentration of lipids in a liposome is a good step in the right direction. Normally one describes a ligand in terms of an on and off rate. Certainly lipids have an affinity for ion channels, but we lack the methodology for effectively measuring lipid channel interactions. Better quantitative lipid binding assays are needed. New mass spec techniques will likely allow for quantitative measurements of lipids in vivo and in vitro. And there is no doubt lipidomics will continue to find ways to improve the quantitative, temporal, and spatial identification of lipids in a membrane.

5.2 Implications on the plasma membrane

The plasma membrane holds thousands of lipids with functions that remain largely a mystery [116]. A catalog of lipid signals appears poised to exert exquisite regulation on membrane proteins perhaps rivaled only by protein phosphorylation. Certainly the phosphodiester bonds in lipids are equally suited for rapid signaling. And lipid acyl chains may be as diverse in function as they are in chemistry. Recognizing low abundant phospholipid signaling molecules as potential ligands for membrane proteins reveals a vast pool of putative effector ligands for cellular signaling.

6. Concluding remarks

The added role of PIP2 activation presented here takes shape from the recent crystallographic Kir structures. The non-specific model of PIP2 activating Kir is gradually making room for a PIP2 site with specificity and ligand like properties. How does a lipid ligand influence its target molecule? It does so just like any other molecule; it binds in a concentration dependent manner to a binding site and elicits a conformational change in the protein. While in hindsight this seems an obvious possibility, the plasma membrane has always been a little mysterious [117] and the understanding of membrane proteins slow in coming. No doubt lipid modulation of proteins is diverse with more surprises yet to come.

Fig. 3.

Fig. 3

Mechanistic comparison of surface charge gating vs. direct lipid gating. A, Non-specific surface charge gates an ion channel through a charge sensor domain (blue). The vertical arrow indicates charge driven movement. B, A lipid-gated channel reversibly binds the signaling lipid PIP2 to allosterically gate the channel. A horizontal arrow indicates PIP2 dissociation from the channel.

Highlights.

Acknowledgments

I thank Andrew S. Hansen for helpful discussion and comments on the manuscript. This work was supported by a Director’s New Innovator Award to SBH (1DP2NS087943-01) from the NIH Common Fund and The National Institute of Neurological Disorders and Stroke (NINDS).

Abbreviations

AA

arachidonic acid

ASIC

acid sensing ion channel

ATP

adenosine triphosphate

BK

big conductance potassium channel

Cav

voltage-dependent calcium channel or VDCC

Ci-VSP

Ciona intestinalis voltage sensitive phosphatase

CoA

coenzyme A

CTD

cytoplasmic domain

C8PIP2

dioctanoyl PIP2

DAG

diacylglycerol

DRM

detergent resistant membrane

ER

endoplasmic reticulum

GIRK

G-protein inward rectifying potassium channel or Kir3

Gβγ

G-protein beta gamma subunit

GPCR

G-protein coupled receptor

HCN

hyperpolarization-activated cyclic nucleotide-gated

IP3

inositol triphosphate

Katp

ATP- sensitive potassium channel or Kir6

Kir

inward rectifying potassium channel

Kv

voltage-gated potassium channel

K2P

two pore domain potassium channel

LAT

lipid acyl transferase

Ld

liquid disordered phase

MARCKS

myristoylated alanine-rich C-kinase substrate

Mg

magnesium

NMDA

N-methyl-D-aspartate receptor

nAChR

nicotinic acetylcholine receptor

PA

phosphatidic acid

PH

pleckstrin homology

PI

phosphoinositide

PIP2

phosphatidylinositol 4,5-bisphosphate

PIP3

phosphatidylinositol 3,4,5-triphosphate

PI3 kinase

phosphatidylinositol-4,5-bisphosphate 3-kinase

PLA2

phospholipase A2

PLC

phospholipase C

PLD

phospholipase D

PS

phosphatidylserine

PTEN

phosphatase and tensin homolog

PUFA

polyunsaturated fatty acid

P2X

purinergic receptors

Sn2

stereospecific numbering position 2 or the second hydroxyl group of glycerol

TMD

transmembrane domain

TM1

transmembrane helix 1

TREK

TWIK related potassium channel or K2P2.1

TRP

transient receptor potential channel

VSD

voltage sensor domain

Footnotes

Conflict of interest.

The author declares no conflict of interest.

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Bibliography