Alcohol Pocket Research Articles

Identification of a G-Protein-Independent Activator of GIRK Channels

G-protein-gated inwardly rectifying K+ (GIRK) channels are essential effectors of inhibitory neurotransmission in the brain. GIRK channels have been implicated in diseases with abnormal neuronal excitability, including epilepsy and addiction. GIRK channels are tetramers composed of either the same subunit (e.g., homotetramers) or different subunits (e.g., heterotetramers). Compounds that specifically target subsets of GIRK channels invivo are lacking. Previous studies have shown that alcohol directly activates GIRK channels through a hydrophobic pocket located in the cytoplasmic domain of the channel. Here, we report the identification and functional characterization of a GIRK1-selective activator, termed GiGA1, that targets the alcohol pocket. GiGA1 activates GIRK1/GIRK2 both invitro and invivo and, in turn, mitigates the effects of a convulsant in an acute epilepsy mouse model. These results shed light on the structure-based development of subunit-specific GIRK modulators that could provide potential treatments for brain disorders.

Small Molecule Modulation of Brain GIRK Channels

G protein-gated inwardly rectifying potassium (GIRK) channels regulate cellular excitability in the heart and brain. Stimulation of G protein-coupled receptors that couple to Gi/o (pertussis toxin-sensitive) G proteins is the primary mechanism of activating GIRK channels. However, there are several small molecule components of GIRK channel activity that operate independently of the G protein-mediated pathway. The lipid environment surrounding GIRK channels in the plasma membrane is one important regulator of GIRK channel activity. Previous studies have established that the membrane phospholipid PIP2 is essential for GIRK channel gating. GIRK channel activators, such as Gβγ and alcohol (ethanol), appear to work by enhancing the interaction with PIP2. We studied the channel gating properties of the brain GIRK2 channel in a defined reconstituted system that allowed for precise control of small molecules and ions. We found that cholesterol, a major component of brain membranes, potentiates the neuronal GIRK2 channel activity in a PIP2 dependent manner. Cholesterol shifts the PIP2 dose-response curve to lower concentrations, demonstrating enhanced PIP2 binding to the channel. Ethanol activates GIRK channels independently of the agonist-mediated pathway via binding to an alcohol pocket in the cytoplasmic terminal domain. There is no apparent shift in alcohol sensitivity in the presence of cholesterol, suggesting alcohol and cholesterol interact structurally and mechanistically with different regions of the channel. Elucidating the molecular mechanism underlying the activation of GIRK channels by small compounds, such as ethanol and cholesterol, should aid in the design of new drugs for treating alcoholism and other neurological disorders.

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site.

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.

Identification of an Inhibitory Alcohol Binding Site in GABAaRho1

We examined why alcohols inhibit the γ-aminobutyric acid type A rho1 receptor (GABAaRrho1) function whereas alcohols potentiate other GABAaR subtypes. We did so by introducing mutations in the second transmembrane helix in GABAaRrho1. Homology models of rho1 receptors based on the X-ray structure of GluCl showed that the 2′, 5′, 6’ and 9′ residues were easily accessible from the ion pore, with 5′ and 6′ residues from neighboring subunits facing each other; whereas L3′ and L277(TM1) were facing the neighboring subunit. We studied ethanol and hexanol effects on GABA responses using two-voltage clamp electrophysiology in Xenopus laevis oocytes. The 6′ mutations showed changes in ethanol and hexanol effects: small changes (T6′M), increased inhibition (T6′V), and small potentiation (T6′Y and T6′F). The 5′ mutations produced increased hexanol inhibition. Other mutations produced smaller (3′ and 9′) or no changes (2′ and L277) on alcohol effects. These results suggest an inhibitory alcohol binding site near the 6′ position By testing ethanol through octanol on single and double mutated rho1 receptors [rho1(I15′S), rho1(T6′Y) and rho1(T6′Y,I15′S)], we further characterized the inhibitory alcohol pocket in the wild-type ρ1 receptor that can only bind relatively short-chain alcohols, and is eliminated by introducing Y in the 6’ position. Replacing the 15′ residue with a smaller side chain introduced a potentiating binding site, more sensitive to long-chain than to short-chain alcohols. In conclusion, the net alcohol effect on the ρ1 receptor is determined by the sum of its actions on inhibitory and potentiating sites.

Identification of an Inhibitory Alcohol Binding Site in GABAA ρ1 Receptors.

Alcohols inhibit γ-aminobutyric acid type A ρ1 receptor function. After introducing mutations in several positions of the second transmembrane helix in ρ1, we studied the effects of ethanol and hexanol on GABA responses using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. The 6' mutations produced the following effects on ethanol and hexanol responses: small increase or no change (T6'M), increased inhibition (T6'V), and small potentiation (T6'Y and T6'F). The 5' mutations produced mainly increases in hexanol inhibition. Other mutations produced small (3' and 9') or no changes (2' and L277 in the first transmembrane domain) in alcohol effects. These results suggest an inhibitory alcohol binding site near the 6' position. Homology models of ρ1 receptors based on the X-ray structure of GluCl showed that the 2', 5', 6', and 9' residues were easily accessible from the ion pore, with 5' and 6' residues from neighboring subunits facing each other; L3' and L277 also faced the neighboring subunit. We tested ethanol through octanol on single and double mutated ρ1 receptors [ρ1(I15'S), ρ1(T6'Y), and ρ1(T6'Y,I15'S)] to further characterize the inhibitory alcohol pocket in the wild-type ρ1 receptor. The pocket can only bind relatively short-chain alcohols and is eliminated by introducing Y in the 6' position. Replacing the bulky 15' residue with a smaller side chain introduced a potentiating binding site, more sensitive to long-chain than to short-chain alcohols. In conclusion, the net alcohol effect on the ρ1 receptor is determined by the sum of its actions on inhibitory and potentiating sites.

Alcohol Inhibition of a Chemically-Activated GIRK2 Channel

Alcohol is widely used and often abused. Yet, the molecular understanding of its action on brain function is poorly understood. Alcohol directly modulates the activity of several ion channels, including the G protein-gated inwardly-rectifying potassium (GIRK) channel. GIRK channels are directly activated by alcohol, independent of their typical G-protein mediated pathway. Currently, however, the molecular mechanism underlying alcohol activation of GIRK is poorly understood. We recently demonstrated that introduction of a Cysteine into the alcohol pocket of GIRK2(L257C) created a channel that could be chemically activated with alcohol-like cysteine-reacting reagents. Here, we studied the channel gating properties of purified GIRK2-L257C channels in a defined reconstituted system that allowed precise control of the lipids, G proteins and ions. We expressed a truncated cys-less GIRK2-L257C(GIRK2Δ∗-L257C) in Pichia pastoris, reconstituted purified protein into liposomes and studied the function of purified GIRK2Δ∗ L257C using a high throughput potassium flux assay. Reconstitution of GIRK2Δ∗-L257C into POPE:POPG:PIP2 containing liposomes exhibited a basal, barium-sensitive flux that was potently enhanced by pre-incubation with MTS-hydroxyethyl(MTS-HE) as well as the Gβγ G-protein subunits. Propanol treatment also enhanced the K+ flux. Thus, in the absence of any other proteins or cytoplasmic regulators, these experiments demonstrate the direct activation of GIRK2 channels by three distinct ligands, Gβγ G-proteins, alcohol and MTS-HE. Interestingly, MTS-HE-activated GIRK2Δ∗-L257C channels were inhibited by propanol in a dose-dependent manner. Inclusion of TCEP (tris(2-carboxyethyl)phosphine), which reduces disulfides, decreased MTS-HE activation but converted the propanol response from inhibition to activation. These experiments reveal that GIRK2 channels can also be inhibited by alcohol, perhaps through a different site, depending on the level of basal channel activation. Elucidating the details underlying alcohol's effects on channel proteins is paramount to developing selective pharmacological tools that could be used in the treatment of alcohol abuse and addiction.

Alcohol modulation of G-protein-gated inwardly rectifying potassium channels: from binding to therapeutics

Alcohol (ethanol)-induced behaviors may arise from direct interaction of alcohol with discrete protein cavities within brain proteins. Recent structural and biochemical studies have provided new insights into the mechanism of alcohol-dependent activation of G protein-gated inwardly rectifying potassium (GIRK) channels, which regulate neuronal responses in the brain reward circuit. GIRK channels contain an alcohol binding pocket formed at the interface of two adjacent channel subunits. Here, we discuss the physiochemical properties of the alcohol pocket and the roles of G protein βγ subunits and membrane phospholipid PIP2 in regulating the alcohol response of GIRK channels. Some of the features of alcohol modulation of GIRK channels may be common to other alcohol-sensitive brain proteins. We discuss the possibility of alcohol-selective therapeutics that block alcohol access to the pocket. Understanding alcohol recognition and modulation of brain proteins is essential for development of therapeutics for alcohol abuse and addiction.

Dissecting Gating Rules of GIRK Channels: Role of PIP2 in Ethanol-Dependent Activation

G protein-gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse and addiction. We discovered a discrete alcohol-binding pocket in the channel mediating ethanol-dependent activation. Here, we investigated the role of G proteins and PIP2 in ethanol-dependent gating. We engineered GIRK2 with single, modifiable cysteine at L257 in alcohol pocket and found that alcohol-like methanthiosulfonate (MTS) reagents activate GIRK2-L257C, similar to ethanol-dependent activation. We assessed role of G proteins in alcohol activation by either increasing levels of Gβγ (+Gβ1γ2) or decreasing Gβγ through chelation with membrane bound Phosducin (+mPhos). Neither Gβ1γ2 nor mPhos altered the rate of MTS-HE-mediated GIRK2-L257C activation. For comparison, we examined GIRK2-L344C, a key site for Gβγ activation that is inhibited by MTS modification. In contrast to L257C, rate of MTS modification showed a clear dependence on Gβγ levels. These results suggest that alcohol activation of GIRK channel is independent of G proteins.To investigate the role of PIP2, we used voltage-sensitive phosphatase DR-VSP to transiently deplete PIP2 in the membrane. Activation of DR-VSP completely reversed MTS-HE activated current of GIRK2-L257C. Furthermore, MTS-HE treatment significantly slowed the rate of GIRK2-L257C current inhibition following DR-VSP activation, suggesting an increase in apparent affinity for PIP2 and GIRK2-L257C channels modified by MTS-HE. Lastly, we examined the role of PIP2 on alcohol-dependent activation of wild-type GIRK2. Addition of propanol significantly slowed the rate of wild-type GIRK2 current inhibition following PIP2 depletion. Taken together, these data demonstrate that alcohol-dependent activation of GIRK involves an increase in apparent affinity for PIP2, with little influence of Gβγ subunits. The fundamental dichotomy between alcohol and Gβγ arises from distinct gating mechanisms converging on PIP2-dependent opening, revealing novel pathways for antagonizing alcohol's effects on ion channels.

Engineering the Alcohol Pocket to Create a Chemically-Activated GIRK Channel

G-protein gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse. Ethanol directly activates GIRKs through interaction with a discrete alcohol-binding pocket that we identified in the cytoplasmic domain. The structural mechanism underlying ethanol-dependent activation, however, is not well understood. Here, we hypothesized that chemically modifying the alcohol pocket with alcohol-like reagents would activate the channel. To examine this, we engineered GIRK2 containing a single cytoplasmic cysteine substitution in the βD-βE domain of the alcohol pocket; S246 is located near the edge of the pocket, and L257 is located within the pocket. All Cys substitutions were introduced into GIRK2c lacking four native Cys residues (GIRK2c(cys-)) and transiently expressed in HEK293T cells. We studied the effect of 2-Hydroxyethyl MTS (MTS-HE, alcohol-like) and two hydrophobic MTS reagents- Benzyl-MTS (MTS-Bn), and 4-hydroxy benzyl-MTS (MTS-Y), on basal (Ba2+-sensitive) and alcohol-activated GIRK currents. MTS-HE (100 μM) increased basal GIRK currents for S246C (34% ± 12%, n=7) and L257C (120% ± 24%, n=7). Similarly, MTS-Bn (10 μM) modification increased basal currents by 74% ± 18% (n=7) for S246C, and by 333% ± 31% (n=4) for L257C. MTS-Y (100 μM) increased the basal currents by 102% ± 25% (n= 5) for L257C but did not affect S246C. In addition, MTS modification decreased MPD-activated current. For L257C, we confirmed that intracellular application of MTS-HE produced a concentration-dependent increase in the time course of channel activation, having a rate constant of 90.6±17.2 M−1s−1 (n=11). Taken together, these data demonstrate that MTS modification of a single amino acid in the alcohol pocket can engage channel activation, and limit the access of bulkier diols (e.g., MPD). These results reveal novel mechanisms for chemical activation of GIRKs and underscore the importance of hydrophobic interactions in alcohol-mediated activation of GIRKs.

Ivermectin Antagonizes Ethanol Inhibition in Purinergic P2X4 Receptors

ATP-gated purinergic P2X4 receptors (P2X4Rs) are expressed in the central nervous system and are sensitive to ethanol at intoxicating concentrations. P2XRs are trimeric; each subunit consists of two transmembrane (TM) alpha-helical segments, a large extracellular domain, and intracellular amino and carboxyl terminals. Recent work indicates that position 336 (Met336) in the TM2 segment is critical for ethanol modulation of P2X4Rs. The anthelmintic medication ivermectin (IVM) positively modulates P2X4Rs and is believed to act in the same region as ethanol. The present study tested the hypothesis that IVM can antagonize ethanol action. We investigated IVM and ethanol effects in wild-type and mutant P2X4Rs expressed in Xenopus oocytes by using a two-electrode voltage clamp. IVM antagonized ethanol-induced inhibition of P2X4Rs in a concentration-dependent manner. The size and charge of substitutions at position 336 affected P2X4R sensitivity to both ethanol and IVM. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed a pocket formed by Asp331, Met336, Trp46, and Trp50 that may play a role in the actions of ethanol and IVM. These findings provide the first evidence for IVM antagonism of ethanol effects in P2X4Rs and suggest that the antagonism results from the ability of IVM to interfere with ethanol action on the putative pocket at or near position 336. Taken with the building evidence supporting a role for P2X4Rs in ethanol intake, the present findings suggest that the newly identified alcohol pocket is a potential site for development of medication for alcohol use disorders.

Identification of the Alcohol Activation Site in GIRK Channels

In addition to G proteins, ethanol can activate G protein-gated inwardly rectifying K (GIRK) channels. The mechanism underlying GIRK channel activation by alcohol is not well understood. Based on a crystal structure of a related IRK1 channel which contains the alcohol (2-methyl,2-4-pentanediol- MPD) bound to a cytoplasmic hydrophobic pocket, we used structure-based mutagenesis and patch-clamp electrophysiology to investigate the role of the homologous alcohol pocket in GIRK2 channels. In HEK293T cells transfected with GIRK2 cDNA, both ethanol and MPD activated GIRK2 channels. Replacing a conserved Leucine (L257) in this pocket with a bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation of GIRK2 channels, suggesting these larger hydrophobic side-chains filled the pocket. Based on structure and functional evidence, we conclude that this hydrophobic pocket is the site for alcohol activation of GIRK channels. We hypothesized that tethering a hydrophobic group near the pocket might mimic alcohol mediated activation of the channel. To test this idea, we introduced a S246C mutation in a Cysteine-less GIRK2 channel and examined the effect of bath applied MTS-Benzene. Application of 10 micromolar MTS-Benzene dramatically increased the size of basal GIRK currents by 336+66% n=5. This rapid activation was reversed by application of reducing agent DTT (10 mM), indicating a disulfide bond had formed. In addition to the change in basal current, MTS modification of S246C channel altered the rank order for alcohol activation -with significantly less activation by the larger alcohol MPD. These results suggest that attachment of a bulky hydrophobic amino acid near the hydrophobic alcohol-binding pocket can produce sustained activation of the channel by associating with the activation site. These experiments provide a launching point to study molecular events at this hydrophobic pocket that lead to activation of GIRK channels.

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 that binds PIP2 strongly. 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 utilizing this hydrophobic alcohol-binding pocket. These results provide a tool for developing therapeutic compounds that could mitigate the effects of alcohol.

Evidence for a Discrete Alcohol Pocket Mediating GIRK Channel Activation

Alcohols can activate G protein-gated inwardly rectifying K (GIRK) channels but the molecular mechanism is not well understood. To investigate the possibility of a physical alcohol pocket located in the cytoplasmic domain of GIRK channels, we used a crystal structure of related IRK1 channel containing a bound alcohol (2-methyl, 2-4-pentanediol - MPD) and structure-based mutagenesis of GIRK2 and GIRK4 channels combined with patch-clamp electrophysiology. In transiently transfected HEK293 cells, both wild-type GIRK2 and GIRK4 channels were activated by 100 mM ethanol, MPD and 1-Propanol. Replacing a conserved Leucine (GIRK2-L257 / GIRK4-L252) in the betaD-betaE region of the cytoplasmic domain with bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation for both GIRK2 and GIRK4 channels. Constitutively open channels, such as IRK1 and GIRK2-PIP2 (engineered to bind PIP2 with high affinity), on the other hand, were inhibited by ethanol, 1-propanol, 1-butanol and MPD. Mutating the homologous Leucine in IRK1 (L257) or in GIRK2-PIP2 channels did not alter the sensitivity to inhibition by these alcohols, suggesting a second site in the channel is involved in inhibition by alcohols. Consistent with this, mutagenesis of the extracellular pore-helix of GIRK4 and GIRK2-PIP2 channels reduced the sensitivity to alcohol-mediated inhibition. Interestingly, mutation of the conserved Leucine (L257/L252) in the betaD-betaE domain also disrupted G protein-dependent activation, suggesting a common mechanism of activation by alcohols and G-proteins. Using our data and an analysis of high-resolution structures of inwardly rectifying K channels, we propose a novel model for alcohol activation of GIRK channels that is mediated by the cytoplasmic hydrophobic alcohol pocket.

Evidence that ethanol acts on a target in Loop 2 of the extracellular domain of α1 glycine receptors

Considerable evidence indicates that ethanol acts on specific residues in the transmembrane domains of glycine receptors (GlyRs). In this study, we tested the hypothesis that the extracellular domain is also a target for ethanol action by investigating the effect of cysteine substitutions at positions 52 (extracellular domain) and 267 (transmembrane domain) on responses to n-alcohols and propyl methanethiosulfonate (PMTS) in alpha1GlyRs expressed in Xenopus oocytes. In support of the hypothesis: (i) The A52C mutation changed ethanol sensitivity compared to WT GlyRs; (ii) PMTS produced irreversible alcohol-like potentiation in A52C GlyRs; and (iii) PMTS binding reduced the n-chain alcohol cutoff in A52C GlyRs. Further studies used PMTS binding to cysteines at positions 52 or 267 to block ethanol action at one site in order to determine its effect at other site(s). In these situations, ethanol caused negative modulation when acting at position 52 and positive modulation when acting at position 267. Collectively, these findings parallel the evidence that established the TM domain as a target for ethanol, suggest that positions 52 and 267 are part of the same alcohol pocket and indicate that the net effect of ethanol on GlyR function reflects the summation of its positive and negative modulatory effects on different targets.

Unmasking of hydrogen tunneling in the horse liver alcohol dehydrogenase reaction by site-directed mutagenesis

Primary and secondary kD/kT and kH/kT kinetic isotope effects have been studied as a probe of hydrogen tunneling in the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase (LADH). In the case of the wild-type enzyme, isotope effects at 25 degrees C do not clearly support hydrogen tunneling; this result is consistent with a reaction rate that is partially limited by the release of product benzaldehyde. The three-dimensional structure for LADH was used to design site-directed mutations in an effort to enhance the rate of the product release step and to "unmask" tunneling. Substitutions that increased the size of the alcohol binding pocket resulted in minor changes in isotope effects. By contrast, reduction in the size of the alcohol binding pocket through substitution at residues 57 and 93, which are in van der Waals contact with bound alcohol substrate, produced a clear demonstration of protium tunneling from the breakdown of the semiclassical relationship between kD/kT and kH/kT isotope effects. The temperature dependence of kD/kT isotope effects has also been pursued, leading to the conclusion that tunneling does, in fact, occur in the reaction catalyzed by wild-type LADH. Despite the unmasking of protium tunneling in site-directed mutants, substitutions that decrease the size of the alcohol pocket appear to result in less extensive tunneling in the hydride transfer. It is noteworthy that the mutant enzyme (Leu57-->Phe), which shows the greatest evidence of tunneling, has the same catalytic efficiency (Vmax/Km) as the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)