Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2.

Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP2 and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.

Dynamic role of the tether helix in PIP2-dependent gating of a G protein-gated potassium channel.

G protein-gated inwardly rectifying potassium (GIRK) channels control neuronal excitability in the brain and are implicated in several different neurological diseases. The anionic phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is an essential cofactor for GIRK channel gating, but the precise mechanism by which PIP2 opens GIRK channels remains poorly understood. Previous structural studies have revealed several highly conserved, positively charged residues in the "tether helix" (C-linker) that interact with the negatively charged PIP2 However, these crystal structures of neuronal GIRK channels in complex with PIP2 provide only snapshots of PIP2's interaction with the channel and thus lack details about the gating transitions triggered by PIP2 binding. Here, our functional studies reveal that one of these conserved basic residues in GIRK2, Lys200 (6'K), supports a complex and dynamic interaction with PIP2 When Lys200 is mutated to an uncharged amino acid, it activates the channel by enhancing the interaction with PIP2 Atomistic molecular dynamic simulations of neuronal GIRK2 with the same 6' substitution reveal an open GIRK2 channel with PIP2 molecules adopting novel positions. This dynamic interaction with PIP2 may explain the intrinsic low open probability of GIRK channels and the mechanism underlying activation by G protein Gßγ subunits and ethanol.

The effects of stretch activation on ionic selectivity of the TREK-2 K2P K+ channel.

The TREK-2 (KCNK10) K2P potassium channel can be regulated by variety of polymodal stimuli including pressure. In a recent study, we demonstrated that this mechanosensitive K+ channel responds to changes in membrane tension by undergoing a major structural change from its 'down' state to the more expanded 'up' state conformation. These changes are mostly restricted to the lower part of the protein within the bilayer, but are allosterically coupled to the primary gating mechanism located within the selectivity filter. However, any such structural changes within the filter also have the potential to alter ionic selectivity and there are reports that some K2Ps, including TREK channels, exhibit a dynamic ionic selectivity. In this addendum to our previous study we have therefore examined whether the selectivity of TREK-2 is altered by stretch activation. Our results reveal that the filter remains stable and highly selective for K+ over Na+ during stretch activation, and that permeability to a range of other cations (Rb+, Cs+ and NH4+) also does not change. The asymmetric structural changes that occur during stretch activation therefore allow the channel to respond to changes in membrane tension without a loss of K+ selectivity.

Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel.

The mechanosensitive two-pore domain (K2P) K+ channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the "down" to "up" conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.

Functional Annotation of Ion Channel Structures by Molecular Simulation.

Ion channels play key roles in cell membranes, and recent advances are yielding an increasing number of structures. However, their functional relevance is often unclear and better tools are required for their functional annotation. In sub-nanometer pores such as ion channels, hydrophobic gating has been shown to promote dewetting to produce a functionally closed (i.e., non-conductive) state. Using the serotonin receptor (5-HT3R) structure as an example, we demonstrate the use of molecular dynamics to aid the functional annotation of channel structures via simulation of the behavior of water within the pore. Three increasingly complex simulation analyses are described: water equilibrium densities; single-ion free-energy profiles; and computational electrophysiology. All three approaches correctly predict the 5-HT3R crystal structure to represent a functionally closed (i.e., non-conductive) state. We also illustrate the application of water equilibrium density simulations to annotate different conformational states of a glycine receptor.

Polymodal activation of the TREK-2 K2P channel produces structurally distinct open states.

The TREK subfamily of two-pore domain (K2P) K(+) channels exhibit polymodal gating by a wide range of physical and chemical stimuli. Crystal structures now exist for these channels in two main states referred to as the "up" and "down" conformations. However, recent studies have resulted in contradictory and mutually exclusive conclusions about the functional (i.e., conductive) status of these two conformations. To address this problem, we have used the state-dependent TREK-2 inhibitor norfluoxetine that can only bind to the down state, thereby allowing us to distinguish between these two conformations when activated by different stimuli. Our results reconcile these previously contradictory gating models by demonstrating that activation by pressure, temperature, voltage, and pH produce more than one structurally distinct open state and reveal that channel activation does not simply involve switching between the up and down conformations. These results also highlight the diversity of structural mechanisms that K2P channels use to integrate polymodal gating signals.

Molecular simulation studies of hydrophobic gating in nanopores and ion channels.

Gating in channels and nanopores plays a key role in regulating flow of ions across membranes. Molecular simulations provide a 'computational microscope' which enables us to examine the physical nature of gating mechanisms at the level of the single channel molecule. Water enclosed within the confines of a nanoscale pore may exhibit unexpected behaviour. In particular, if the molecular surfaces lining the pore are hydrophobic this promotes de-wetting of the pore. De-wetting is observed as stochastic liquid-vapour transitions within a pore, and may lead to functional closure of a pore to the flow of ions and/or water. Such behaviour was first observed in simulations of simple model nanopores and referred to as 'hydrophobic gating'. Simulations of both the nicotinic acetylcholine receptor and of TWIK-1 potassium channels (the latter alongside experimental studies) suggest hydrophobic gating may occur in some biological ion channels. Current studies are focused on designing hydrophobic gates into biomimetic nanopores.

K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac.

TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.

Influence of lipids on the hydrophobic barrier within the pore of the TWIK-1 K2P channel.

Several recent ion channel structures have revealed large side portals, or 'fenestrations' at the interface between their transmembrane helices that potentially expose the ion conduction pathway to the lipid core of the bilayer. In a recent study we demonstrated that functional activity of the TWIK-1 K2P channel is influenced by the presence of hydrophobic residues deep within the inner pore. These residues are located near the fenestrations in the TWIK-1 structure and promote dewetting of the pore by forming a hydrophobic barrier to ion conduction. During our previous MD simulations, lipid tails were observed to enter these fenestrations. In this addendum to that study, we investigate lipid contribution to the dewetting process. Our results demonstrate that lipid tails from both the upper and lower leaflets can occupy the fenestrations and partially penetrate into the pore. The lipid tails do not sterically occlude the pore, but there is an inverse correlation between the presence of water within the hydrophobic barrier and the number of lipids tails within the lining of the pore. However, dewetting still occurs in the absence of lipids tails, and pore hydration appears to be determined primarily by those side-chains lining the narrowest part of the pore cavity.

Hydrophobic gating in ion channels.

Biological ion channels are nanoscale transmembrane pores. When water and ions are enclosed within the narrow confines of a sub-nanometer hydrophobic pore, they exhibit behavior not evident from macroscopic descriptions. At this nanoscopic level, the unfavorable interaction between the lining of a hydrophobic pore and water may lead to stochastic liquid-vapor transitions. These transient vapor states are "dewetted", i.e. effectively devoid of water molecules within all or part of the pore, thus leading to an energetic barrier to ion conduction. This process, termed "hydrophobic gating", was first observed in molecular dynamics simulations of model nanopores, where the principles underlying hydrophobic gating (i.e., changes in diameter, polarity, or transmembrane voltage) have now been extensively validated. Computational, structural, and functional studies now indicate that biological ion channels may also exploit hydrophobic gating to regulate ion flow within their pores. Here we review the evidence for this process and propose that this unusual behavior of water represents an increasingly important element in understanding the relationship between ion channel structure and function.

A hydrophobic barrier deep within the inner pore of the TWIK-1 K2P potassium channel.

Recent X-ray crystal structures of the two-pore domain (K2P) family of potassium channels have revealed a unique structural architecture at the point where the cytoplasmic bundle-crossing gate is found in most other tetrameric K(+) channels. However, despite the apparently open nature of the inner pore in the TWIK-1 (K2P1/KCNK1) crystal structure, the reasons underlying its low levels of functional activity remain unclear. In this study, we use a combination of molecular dynamics simulations and functional validation to demonstrate that TWIK-1 possesses a hydrophobic barrier deep within the inner pore, and that stochastic dewetting of this hydrophobic constriction acts as a major barrier to ion conduction. These results not only provide an important insight into the mechanisms which control TWIK-1 channel activity, but also have important implications for our understanding of how ion permeation may be controlled in similar ion channels and pores.

Pharmacological blockade of the cold receptor TRPM8 attenuates autonomic and behavioral cold defenses and decreases deep body temperature.

We studied N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride (M8-B), a selective and potent antagonist of the transient receptor potential melastatin-8 (TRPM8) channel. In vitro, M8-B blocked cold-induced and TRPM8-agonist-induced activation of rat, human, and murine TRPM8 channels, including those on primary sensory neurons. In vivo, M8-B decreased deep body temperature (T(b)) in Trpm8(+/+) mice and rats, but not in Trpm8(-/-) mice, thus suggesting an on-target action. Intravenous administration of M8-B was more effective in decreasing T(b) in rats than intrathecal or intracerebroventricular administration, indicating a peripheral action. M8-B attenuated cold-induced c-Fos expression in the lateral parabrachial nucleus, thus indicating a site of action within the cutaneous cooling neural pathway to thermoeffectors, presumably on sensory neurons. A low intravenous dose of M8-B did not affect T(b) at either a constantly high or a constantly low ambient temperature (T(a)), but the same dose readily decreased T(b) if rats were kept at a high T(a) during the M8-B infusion and transferred to a low T(a) immediately thereafter. These data suggest that both a successful delivery of M8-B to the skin (high cutaneous perfusion) and the activation of cutaneous TRPM8 channels (by cold) are required for the hypothermic action of M8-B. At tail-skin temperatures <23°C, the magnitude of the M8-B-induced decrease in T(b) was inversely related to skin temperature, thus suggesting that M8-B blocks thermal (cold) activation of TRPM8. M8-B affected all thermoeffectors studied (thermopreferendum, tail-skin vasoconstriction, and brown fat thermogenesis), thus suggesting that TRPM8 is a universal cold receptor in the thermoregulation system.

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms.

Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.

A discrete alcohol pocket involved in GIRK channel activation.

Ethanol modifies neural activity in the brain by modulating ion channels. Ethanol activates G protein-gated inwardly rectifying K(+) channels, but the molecular mechanism is not well understood. Here, we used a crystal structure of a mouse inward rectifier containing a bound alcohol and structure-based mutagenesis to probe a putative alcohol-binding pocket located in the cytoplasmic domains of GIRK channels. Substitutions with bulkier side-chains in the alcohol-binding pocket reduced or eliminated activation by alcohols. By contrast, alcohols inhibited constitutively open channels, such as IRK1 or GIRK2 engineered to strongly bind PIP(2). Mutations in the hydrophobic alcohol-binding pocket of these channels had no effect on alcohol-dependent inhibition, suggesting an alternate site is involved in inhibition. Comparison of high-resolution structures of inwardly rectifying K(+) channels suggests a model for activation of GIRK channels using this hydrophobic alcohol-binding pocket. These results provide a tool for developing therapeutic compounds that could mitigate the effects of alcohol.

Evidence for association of GABA(B) receptors with Kir3 channels and regulators of G protein signalling (RGS4) proteins.

Many neurotransmitters and hormones signal by stimulating G protein-coupled neurotransmitter receptors (GPCRs), which activate G proteins and their downstream effectors. Whether these signalling proteins diffuse freely within the plasma membrane is not well understood. Recent studies have suggested that direct protein-protein interactions exist between GPCRs, G proteins and G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels. Here, we used fluorescence resonance energy transfer (FRET) combined with total internal reflection fluorescence microscopy to investigate whether proteins within this signalling pathway move within 100 A of each other in the plasma membrane of living cells. GABA(B) R1 and R2 receptors, Kir3 channels, Galphao subunits and regulators of G protein signalling (RGS4) proteins were each fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) and first assessed for functional expression in HEK293 cells. The presence of the fluorophore did not significantly alter the signalling properties of these proteins. Possible FRET was then investigated for different protein pair combinations. As a positive control, FRET was measured between tagged GABA(B) R1 and R2 subunits ( approximately 12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABA(B) R1 or R2 subunits ( approximately 13% FRET), and between Galphao and GABA(B) R1 or R2 subunits ( approximately 10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABA(B) R1 or R2 subunits ( approximately 10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Galphao. These data are discussed in terms of a model in which GABA(B) receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.

Neurotransmitter acetylcholine negatively regulates neuromuscular synapse formation by a Cdk5-dependent mechanism.

Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.

Overexpression of mitogen-activated protein kinase kinase 6 in the heart improves functional recovery from ischemia in vitro and protects against myocardial infarction in vivo.

The mitogen-activated protein kinases (MAPK) have been the subject of many studies to identify signaling pathways that promote cell survival or death. In cultured cardiac myocytes, p38 MAPK promotes cell survival or death depending on whether it is activated by mitogen-activated protein kinase kinase 6 (MKK6) or MKK3, respectively. The objectives of the current study were to examine the effects of MKK6-mediated p38 activation in the heart in vivo. Accordingly, we generated transgenic (TG) mice that overexpress wild type MKK6 in a cardiac-restricted manner. Although p38 was about 17-fold more active in TG than non-transgenic (NTG) mouse hearts, TG mouse hearts were morphologically and functionally similar to those of NTG littermates. However, upon transient ischemia followed by reperfusion, the MKK6 TG mouse hearts exhibited significantly better functional recovery and less injury than NTG mouse hearts. Because MKK6 increases levels of the protective small heat shock protein, alpha B-crystallin (alpha BC), in cultured cardiac myocytes, we examined alpha BC levels in the mouse hearts. The level of alpha BC was 2-fold higher in MKK6 TG than NTG mouse hearts. Moreover, ischemia followed by reperfusion induced a 6.4-fold increase in alpha BC levels in the mitochondrial fractions of TG mouse hearts but no increase in alpha BC levels in any of the other fractions analyzed. These alterations in alpha BC expression and localization suggest possible mechanisms of cardioprotection in MKK6 TG mouse hearts.

Regulation of myosin-VI targeting to endocytic compartments.

Myosin-VI has been implicated in endocytic trafficking at both the clathrin-coated and uncoated vesicle stages. The identification of alternative splice forms led to the suggestion that splicing defines the vesicle type to which myosin-VI is recruited. In contrast to this hypothesis, we find that in all cell types examined, myosin-VI is associated with uncoated endocytic vesicles, regardless of splice form. GIPC, a PDZ-domain containing adapter protein, co-assembles with myosin-VI on these vesicles. Myosin-VI is only recruited to clathrin-coated vesicles in cells that express high levels of Dab2, a clathrin-binding adapter protein. Overexpression of Dab2 is sufficient to reroute myosin-VI to clathrin-coated pits in cells where myosin-VI is normally associated with uncoated vesicles. In normal rat kidney (NRK) cells, which express high endogenous levels of Dab2, splicing of the globular tail domain further modulates targeting of ectopically expressed myosin-VI. Although myosin-VI can be recruited to clathrin-coated pits, we find no requirement for myosin-VI motor activity in endocytosis in NRK cells. Instead, our data suggest that myosin-VI recruitment to clathrin-coated pits may be an early step in the recruitment of GIPC to the vesicle surface.

Alterations in mitochondrial function in a mouse model of hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by varying degrees of ventricular hypertrophy and myofibrillar disarray. Mutations in cardiac contractile proteins cause HCM. However, there is an unexplained wide variability in the clinical phenotype, and it is likely that there are multiple contributing factors. Because mitochondrial dysfunction has been described in heart disease, we tested the hypothesis that mitochondrial dysfunction contributes to the varying HCM phenotypes. Mitochondrial function was assessed in two transgenic models of HCM: mice with a mutant myosin heavy chain gene (MyHC) or with a mutant cardiac troponin T (R92Q) gene. Despite mitochondrial ultrastructural abnormalities in both models, the rate of state 3 respiration was significantly decreased only in the mutant MyHC mice by approximately 23%. Notably, this decrease in state 3 respiration preceded hemodynamic dysfunction. The maximum activity of alpha-ketogutarate dehydrogenase as assayed in isolated disrupted mitochondria was decreased by 28% compared with isolated control mitochondria. In addition, complexes I and IV were decreased in mutant MyHC transgenic mice. Inhibition of beta-adrenergic receptor kinase, which is elevated in mutant MyHC mouse hearts, can prevent mitochondrial respiratory impairment in mutant MyHC mice. Thus our results suggest that mitochondria may contribute to the hemodynamic dysfunction seen in some forms of HCM and offer a plausible mechanism responsible for some of the heterogeneity of the disease phenotypes.