Search

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future.

β-Secretase 1 (β-site amyloid precursor protein [APP]-cleaving enzyme 1, BACE1) plays a crucial role in the amyloidogenesis of Alzheimer’s disease (AD). BACE1 was also discovered to act like an auxiliary subunit to modulate neuronal KCNQ2/3 channels independently of its proteolytic function. BACE1 is palmitoylated at its carboxyl-terminal region, which brings BACE1 to ordered, cholesterol-rich membrane microdomains (lipid rafts). However, the physiological consequences of this specific localization of BACE1 remain elusive. Using spectral Förster resonance energy transfer (FRET), BACE1 and KCNQ2/3 channels were confirmed to form a signaling complex, a phenomenon that was relatively independent of the palmitoylation of BACE1. Nevertheless, palmitoylation of BACE1 was required for recruitment of KCNQ2/3 channels to lipid-raft domains. Two fluorescent probes, designated L10 and S15, were used to label lipid-raft and non-raft domains of the plasma membrane, respectively. Coexpressing BACE1 substantially elevated FRET between L10 and KCNQ2/3, whereas the BACE1-4C/A quadruple mutation failed to produce this effect. In contrast, BACE1 had no significant effect on FRET between S15 probes and KCNQ2/3 channels. A reduction of BACE1-dependent FRET between raft-targeting L10 probes and KCNQ2/3 channels by applying the cholesterol-extracting reagent methyl-β-cyclodextrin (MβCD), raft-disrupting general anesthetics, or pharmacological inhibitors of palmitoylation, all supported the hypothesis of the palmitoylation-dependent and raft-specific localization of KCNQ2/3 channels. Furthermore, mutating the four carboxyl-terminal cysteines (4C/A) of BACE1 abolished the BACE1-dependent increase of FRET between KCNQ2/3 and the lipid raft–specific protein caveolin 1. Taking these data collectively, we propose that the AD-related protein BACE1 underlies the localization of a neuronal potassium channel.

Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo an inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removal of cAMP produced a largely rigid-body rotation of the C-linker relative to transmembrane domain, bringing the A’ helix in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not in the presence of cAMP. These results suggest the A’ helix functions like a mechanical clutch to engage inactivation. cAMP binding disengages the clutch, permitting the hyperpolarization-dependent activation mediated by the S5 helix. Furthermore, rearrangement of A’ helix during recovery from inactivation of spHCN channels was similar to that for the activation of KCNH channels, suggesting a conserved mechanism for this noncanonical electromechanical coupling in the CNBD channel family.

β-secretase 1 (β-site amyloid precursor protein (APP)-cleaving enzyme 1, BACE1) plays a crucial role in the amyloidogenesis of Alzheimer’s Disease (AD). BACE1 was also discovered to act like an auxiliary subunit to modulate neuronal KCNQ2/3 channels independent of its proteolytic function. BACE1 is palmitoylated at its carboxyl-terminal region, which brings BACE1 to ordered, cholesterol-rich membrane microdomains (lipid rafts). However, the physiological consequences of this specific localization of BACE1 remain elusive. Using spectral Förster Resonance Energy Transfer (FRET), BACE1 and KCNQ2/3 channels were confirmed to form a signaling complex, a phenomenon that was relatively independent of the palmitoylation of BACE1. Nevertheless, palmitoylation of BACE1 was required for recruitment of KCNQ2/3 channels to lipid-raft domains. Two fluorescent probes designated L10 and S15, were used to label lipid-raft and non-raft domains of the plasma membrane, respectively. Coexpressing BACE1 substantially elevated the FRET between L10 and KCNQ2/3 whereas the BACE1-4C/A quadruple mutation failed to produce this effect. In contrast, BACE1 had no significant effect on the FRET between S15 probes and KCNQ2/3 channels. A reduction of BACE1-dependent FRET between raft-targeting L10 probes and KCNQ2/3 channels by applying cholesterol-extracting reagent methyl-β-cyclodextrin (MβCD), raft-disrupting general anesthetics, or pharmacological inhibitors of palmitoylation all supported our hypothesis of the palmitoylation-dependent and raft-specific localization of KCNQ2/3 channels. Furthermore, mutating the four carboxyl-terminal cysteines (4C/A) of BACE1 abolished the BACE1-dependent increase of FRET between KCNQ2/3 and a lipid raft-specific protein caveolin 1. Collectively, we propose how the AD-related protein BACE1 underlies the localization of a neuronal potassium channel.

KCNH voltage-gated potassium channels (EAG, ERG, and ELK) play significant roles in neuronal and cardiac excitability. They contain cyclic nucleotide-binding homology domains (CNBHDs) but are not directly regulated by cyclic nucleotides. Instead, the CNBHD ligand-binding cavity is occupied by an intrinsic ligand, which resides at the intersubunit interface between the N-terminal eag domain and the C-terminal CNBHD. We show that, in Danio rerio ELK channels, this intrinsic ligand is critical for voltage-dependent potentiation (VDP), a process in which channel opening is stabilized by prior depolarization. We demonstrate that an exogenous peptide corresponding to the intrinsic ligand can bind to and regulate zebrafish ELK channels. This exogenous intrinsic ligand inhibits the channels before VDP and potentiates the channels after VDP. Furthermore, using transition metal ion fluorescence resonance energy transfer and a fluorescent noncanonical amino acid L-Anap, we show that there is a rearrangement of the intrinsic ligand relative to the CNBHD during VDP. We propose that the intrinsic ligand switches from antagonist to agonist as a result of a rearrangement of the eag domain–CNBHD interaction during VDP.

Hilgemann et al. explain how lipid signaling to membrane proteins involves a hierarchy of mechanisms from lipid binding to membrane domain coalescence., Lipids influence powerfully the function of ion channels and transporters in two well-documented ways. A few lipids act as bona fide second messengers by binding to specific sites that control channel and transporter gating. Other lipids act nonspecifically by modifying the physical environment of channels and transporters, in particular the protein–membrane interface. In this short review, we first consider lipid signaling from this traditional viewpoint, highlighting innumerable Journal of General Physiology publications that have contributed to our present understanding. We then switch to our own emerging view that much important lipid signaling occurs via the formation of membrane domains that influence the function of channels and transporters within them, promote selected protein–protein interactions, and control the turnover of surface membrane.

Phosphoinositides are rapidly turning-over phospholipids that play key roles in intracellular signaling and modulation of membrane effectors. Through technical refinements we have improved sensitivity in the analysis of the phosphoinositide PI, PIP, and PIP2 pools from living cells using mass spectrometry. This has permitted further resolution in phosphoinositide lipidomics from cell cultures and small samples of tissue. The technique includes butanol extraction, derivatization of the lipids, post-column infusion of sodium to stabilize formation of sodiated adducts, and electrospray ionization mass spectrometry in multiple reaction monitoring mode, achieving a detection limit of 20 pg. We describe the spectrum of fatty-acyl chains in the cellular phosphoinositides. Consistent with previous work in other mammalian primary cells, the 38:4 fatty-acyl chains dominate in the phosphoinositides of the pineal gland and of superior cervical ganglia, and many additional fatty acid combinations are found at low abundance. However, Chinese hamster ovary cells and human embryonic kidney cells (tsA201) in culture have different fatty-acyl chain profiles that change with growth state. Their 38:4 lipids lose their dominance as cultures approach confluence. The method has good time resolution and follows well the depletion in < 20 s of both PIP2 and PIP that results from strong activation of Gq-coupled receptors. The receptor-activated phospholipase C exhibits no substrate selectivity among the various fatty-acyl chain combinations.

EAG-like (ELK) voltage-gated potassium channels are abundantly expressed in the brain. These channels exhibit a behavior called voltage-dependent potentiation (VDP), which appears to be a specialization to dampen the hyperexitability of neurons. VDP manifests as a potentiation of current amplitude, hyperpolarizing shift in voltage sensitivity, and slowing of deactivation in response to a depolarizing prepulse. Here we show that VDP of D. rerio ELK channels involves the structural interaction between the intracellular N-terminal eag domain and C-terminal CNBHD. Combining transition metal ion FRET, patch-clamp fluorometry, and incorporation of a fluorescent noncanonical amino acid, we show that there is a rearrangement in the eag domain-CNBHD interaction with the kinetics, voltage-dependence, and ATP-dependence of VDP. We propose that the activation of ELK channels involves a slow open-state dependent rearrangement of the direct interaction between the eag domain and CNBHD, which stabilizes the opening of the channel. DOI: http://dx.doi.org/10.7554/eLife.26355.001, eLife digest In humans and other animals, electrical signals trigger the heart to beat and carry information around the brain and nervous system. Particular cells can generate these signals by regulating the flow of ions into and out of the cell via proteins called ion channels. These proteins sit in the membrane that surrounds the cell and will open or close in response to specific signals. For example, an ion channel in humans called hERG allows positively-charged potassium ions to flow out of a heart cell to help the cell return to its “resting” state after producing an electrical signal. Defects in hERG can alter the rhythm at which the heart beats, leading to a serious condition called Long QT syndrome. The human hERG channel is part of a family of related channels known as the KCNH channels. These channels are made of four protein subunits that assemble to form a pore that spans the cell membrane. When a cell is resting before producing an electrical signal, KCNH channels are generally closed. However, once an electrical signal starts, the flow of ions through other ion channels in the cell membrane changes an electrical property across the membrane known as the “voltage”. This change in voltage causes KCNH channels to open. Dai and Zagotta studied how a KCNH channel known as ELK from zebrafish responds to changes in membrane voltage. The experiments show that the manner in which ELK channels respond to the voltage is due to changes in how the subunits interact in the part of the channel that lies inside the cell. Further experiments using several new techniques reveal in much more detail how the shape of the channel alters as the voltage changes. These new techniques could also be used to observe how other KCNH channels in the heart and brain change shape in response to changes in voltage. This could lead to the design of new drugs to treat heart and neurological diseases. DOI: http://dx.doi.org/10.7554/eLife.26355.002

Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ∼2-fold greater shift in K1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIPn) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIPn sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIPn regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIPn sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIPn. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides.

Myo-inositol is an important cellular osmolyte in autoregulation of cell volume and fluid balance, particularly for mammalian brain and kidney cells. We find it also regulates excitability. Myo-inositol is the precursor of phosphoinositides, key signaling lipids including phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. However, whether myo-inositol accumulation during osmoregulation affects signaling and excitability has not been fully explored. We found that overexpression of the Na(+)/myo-inositol cotransporter (SMIT1) and myo-inositol supplementation enlarged intracellular PI(4,5)P2 pools, modulated several PI(4,5)P2-dependent ion channels including KCNQ2/3 channels, and attenuated the action potential firing of superior cervical ganglion neurons. Further experiments using the rapamycin-recruitable phosphatase Sac1 to hydrolyze PI(4)P and the P4M probe to visualize PI(4)P suggested that PI(4)P levels increased after myo-inositol supplementation with SMIT1 expression. Elevated relative levels of PIP and PIP2 were directly confirmed using mass spectrometry. Inositol trisphosphate production and release of calcium from intracellular stores also were augmented after myo-inositol supplementation. Finally, we found that treatment with a hypertonic solution mimicked the effect we observed with SMIT1 overexpression, whereas silencing tonicity-responsive enhancer binding protein prevented these effects. These results show that ion channel function and cellular excitability are under regulation by several "physiological" manipulations that alter the PI(4,5)P2 setpoint. We demonstrate a previously unrecognized linkage between extracellular osmotic changes and the electrical properties of excitable cells.

EAG-like (ELK, Kv12) voltage-gated potassium channels in the KCNH channel family are primarily and abundantly expressed in the brain. Deletion of the ELK gene leads to hyperexcitability of hippocampal neurons, epilepsy and altered cognitive functions. One structural feature of KCNH channels important for channel function is an interaction between the N-terminal EAG domain and the C-terminal cyclic nucleotide-binding homology domain (CNBHD). Here we studied an orthologous zebrafish ELK channel (zELK) using patch-clamp recording and patch-clamp fluorometry (PCF). We noticed that a depolarizing prepulse potentiated the zELK channels in both the current amplitude and the voltage sensitivity. We named this phenomenon voltage-dependent potentiation (VDP) and found it developed and recovered within hundreds of milliseconds. Using direct application of diC8-PI(4,5)P2 and specific hydrolysis of PI(4,5)P2 by rapamycin-recruitable lipid 5-phosphatase, we demonstrated that, PI(4,5)P2 inhibits zELK channels, and its depletion suppresses the VDP. In addition, the VDP can be manipulated by multiple structural perturbations including removing, mutating or replacing the intracellular N-terminal EAG domain as well as the C-terminal CNBHD. Further, combining transition metal ion FRET and incorporation of a fluorescent noncanonical amino acid L-Anap, we measured the distances between positions in the EAG domain and CNBHD during voltage-dependent channel activation and deactivation. We found there was an apparent change in the distance between the EAG domain and CNBHD with kinetics that match the on- and off-rates of VDP. This change in the distance between the N- and C-terminal regions was abolished when PI(4,5)P2 was hydrolyzed, and the simultaneous VDP was eliminated. Collectively, we propose that the activation of zELK channels involves a rearrangement of the direct interaction between the N- and C-terminal regions, which transitions the channel to a potentiated state.

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation.

Cyclic nucleotide-gated (CNG) channels in retinal photoreceptors play a crucial role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric cone CNGA3 (A3) + CNGB3 (B3) channels are inhibited by membrane phosphoinositides (PIPn), including phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), demonstrating a decrease in apparent affinity for cyclic guanosine monophosphate (cGMP). Unlike homomeric A1 or A2 channels, A3-only channels paradoxically did not show a decrease in apparent affinity for cGMP after PIPn application. However, PIPn induced an ∼2.5-fold increase in cAMP efficacy for A3 channels. The PIPn-dependent change in cAMP efficacy was abolished by mutations in the C-terminal region (R643Q/R646Q) or by truncation distal to the cyclic nucleotide-binding domain (613X). In addition, A3-613X unmasked a threefold decrease in apparent cGMP affinity with PIPn application to homomeric channels, and this effect was dependent on conserved arginines within the N-terminal region of A3. Together, these results indicate that regulation of A3 subunits by phosphoinositides exhibits two separable components, which depend on structural elements within the N- and C-terminal regions, respectively. Furthermore, both N and C regulatory modules in A3 supported PIPn regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIPn sensitivity to heteromeric channels formed with PIPn-insensitive A subunits. Finally, channels formed by mixtures of PIPn-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIPn regulation, implying that intersubunit N–C interactions help control the phosphoinositide sensitivity of cone CNG channels.

Cyclic nucleotide-gated (CNG) ion channels regulate the electrical activity of retinal photoreceptors, rods and cones, by sensing the light-induced changes of intracellular cGMP levels. Cone CNG channels consist of CNGA3 and modulatory CNGB3 subunits, both of which contain a cyclic nucleotide-binding domain (CNBD). CNGB3 subunits confer enhanced responses to cAMP and support several aspects of channel regulation. However, it is not fully understood how CNGB3 (and CNGA3) are specialized to contribute to ligand-specific activation of cone CNG channels. using patch-clamp recordings, we characterized several mutations located within the CNBD of CNGA3, each of which produced dramatic, ligand-specific effects on channel gating. In particular, D609M in CNGA3 reversed ligand selectivity, making cAMP a better agonist than cGMP, similar to equivalent mutations in paralogous channel subunits. These experiments suggest that mechanisms underlying ligand interaction with CNGA3 are well conserved. However, parallel mutations within the CNBD Cα-helix of CNGB3 had no effect on the ligand selectivity of heteromeric channels, consistent with the large decrement in sequence conservation in this region of CNGB3. CNGB3 appear to lack features supporting ligand discrimination. Next, we examined subunit contributions to ligand-dependent activation using CNBD “knock out” (R564E in CNGA3; R604E in CNGB3). CNGB3 R604E decreased relative cAMP efficacy, but only had a subtle effect on the cGMP activation for heteromeric channels (with wild-type or R564E CNGA3). In contrast, CNGA3 R564E caused an approximately 500-fold decrease in apparent cGMP affinity and nearly eliminated cAMP-dependent gating. Similar results were observed with analogous experiments using mutations of T565A in CNGA3 and T605A in CNGB3. Together, we propose that CNGA3 is the principal subunit mediating both ligand discrimination and ligand-dependent stabilization of the open state, while CNGB3 makes only a minor contribution to cGMP-dependent gating.

Precursor mRNA encoding CNGA3 subunits of cone photoreceptor CNG channels undergoes alternative splicing, generating variants differing in the N-terminal cytoplasmic region of the protein. In humans, four CNGA3 protein variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Because CNGA3 subunits mediate cone CNG channel regulation by phosphoinositides (PIs), we hypothesized that alternative splicing may tune channel sensitivity to PIs. Heterologous expression of the four CNGA3 variants alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon e3, with or without optional exon e5, produced heteromeric CNGA3+CNGB3 channels exhibiting an approximately three-fold greater shift in K1/2,cGMP after PIP2 or PIP3 compared to channels lacking the sequence encoded by exon e3. The relative abundance of exon e3-containing retinal transcripts was ∼20% by qPCR. We have previously identified two structural features within CNGA3 that support PI regulation of cone CNG channels: an N-terminal and a C-terminal component; specific mutations within these regions eliminated PI sensitivity of CNGA3+CNGB3 channels. The e3 variant enhanced the component of PI modulation that depends on the C-terminal region, rather than the nearby N-terminal region, consistent with an allosteric effect on PI sensitivity. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon e3 is unique to humans, even compared to other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region that is necessary for high sensitivity to PIs. Presumably, alternative splicing in CNGA3 has evolved to regulate the interactions between the channel and membrane-bound phospholipids. Together, these results reveal an important mechanism of channel control that may have broad implications.