Brett A. Cromer

Anxiety over GABAA receptor structure relieved by AChBP

The GABA(A) receptor is the primary mediator of inhibitory neurotransmission in the brain and is a major target for neuromodulatory drugs such as benzodiazepines, barbiturates, ethanol and anaesthetics. However, our understanding of the molecular details of this receptor has been limited by a lack of high-resolution structural information. This article presents a new model for the extracellular, ligand-binding domain of the GABA(A) receptor, that is based on the recently determined structure of a soluble acetylcholine-binding protein. The model puts existing mutational and biochemical data into a three-dimensional context, shows details of the GABA- and benzodiazepine-binding sites, and highlights the importance of other regions in allosteric conformational change. This provides a new perspective on existing data and an exciting new framework for understanding this important family of receptors.

Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein.

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5–7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5–7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.

Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit mutation [γ2(R43Q)] found in human epilepsy

The γ-aminobutyric acid type A (GABAA) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABAA receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABAA γ2-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABAA receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from α1β2γ2 or α1β2γ2(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imidazopyridine zolpidem. These results provide evidence of impaired GABAA receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

From glutathione transferase to pore in a CLIC.

Abstract. Many plasma membrane chloride channels have been cloned and characterized in great detail. In contrast, very little is known about intracellular chloride channels. Members of a novel class of such channels, called the CLICs (chloride intracellular channels), have been identified over the last few years. A striking feature of the CLIC family of ion channels is that they can exist in a water-soluble state as well as a membrane-bound state. A major step forward in understanding the functioning of these channels has been the recent crystal structure determination of one family member, CLIC1. The structure confirms that CLICs are members of the glutathione S-transferase superfamily and provides clues as to how CLICs can insert into membranes to form chloride channels.

Conductance of recombinant GABA (A) channels is increased in cells co-expressing GABA(A) receptor-associated protein

High conductance γ-aminobutyric acid type A (GABAA) channels (>40 picosiemens (pS)) have been reported in some studies on GABAA channels in situ but not in others, whereas recombinant GABAA channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABAA channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABAA channels. GABARAP, a protein associated with native GABAA channels, has been reported to cause clustering of GABAA receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing α1, β1, and γ2S subunits of human GABAA receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABAA receptors that make them open co-operatively to give high conductance “channels.” Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.

Augmented currents of an HCN2 variant in patients with febrile seizure syndromes

The genetic architecture of common epilepsies is largely unknown. HCNs are excellent epilepsy candidate genes because of their fundamental neurophysiological roles. Screening in subjects with febrile seizures and genetic epilepsy with febrile seizures plus revealed that 2.4% carried a common triple proline deletion (delPPP) in HCN2 that was seen in only 0.2% of blood bank controls. Currents generated by mutant HCN2 channels were ∼35% larger than those of controls; an effect revealed using automated electrophysiology and an appropriately powered sample size. This is the first association of HCN2 and familial epilepsy, demonstrating gain of function of HCN2 current as a potential contributor to polygenic epilepsy. ANN NEUROL 2010;67:542–546

INHIBITION OF SKELETAL MUSCLE ClC-1 CHLORIDE CHANNELS BY LOW INTRACELLULAR pH AND ATP

Skeletal muscle acidosis during exercise has long been thought to be a cause of fatigue, but recent studies have shown that acidosis maintains muscle excitability and opposes fatigue by decreasing the sarcolemmal chloride conductance. ClC-1 is the primary sarcolemmal chloride channel and has a clear role in controlling muscle excitability, but recombinant ClC-1 has been reported to be activated by acidosis. Following our recent finding that intracellular ATP inhibits ClC-1, we investigated here the interaction between pH and ATP regulation of ClC-1. We found that, in the absence of ATP, intracellular acidosis from pH 7.2 to 6.2 inhibited ClC-1 slightly by shifting the voltage dependence of common gating to more positive potentials, similar to the effect of ATP. Importantly, the effects of ATP and acidosis were cooperative, such that ATP greatly potentiated the effect of acidosis. Adenosine had a similar effect to ATP at pH 7.2, but acidosis did not potentiate this effect, indicating that the phosphates of ATP are important for this cooperativity, possibly due to electrostatic interactions with protonatable residues of ClC-1. A protonatable residue identified by molecular modeling, His-847, was found to be critical for both pH and ATP modulation and may be involved in such electrostatic interactions. These findings are now consistent with, and provide a molecular explanation for, acidosis opposing fatigue by decreasing the chloride conductance of skeletal muscle via inhibition of ClC-1. The modulation of ClC-1 by ATP is a key component of this molecular mechanism.

A proposed structural basis for picrotoxinin and picrotin binding in the glycine receptor pore

Picrotoxin, an antagonist of structurally‐rated GABAA receptors (GABAARs) and glycine receptors (GlyRs), is an equimolar mixture of picrotoxinin (PTXININ) and picrotin (PTN). These compounds share a common structure except that PTN contains a slightly larger dimethylmethanol in place of the PTXININ isopropenyl group. Although the homomeric α1 GlyR is equally sensitive to both compounds, we show here that homomeric α2 and α3 GlyRs, like most GABAARs, are selectively inhibited by PTXININ. As conservative mutations to pore‐lining 6′ threonines equally affect the sensitivity of the α1 GlyR to both compounds, we conclude that PTXININ and PTN bind to 6′ threonines by hydrogen bonding with exocyclic oxygens common to both molecules. In contrast, substitution of the 2′ pore‐lining glycine by serine selectively reduces PTN sensitivity, whereas the introduction of 2′ alanines selectively increases PTXININ sensitivity. These results define the orientation of PTXININ and PTN binding in the α1 GlyR pore and allow us to conclude that the relatively reduced sensitivity of PTN at GABAARs and α2 and α3 GlyRs is due predominantly to its larger size and reduced ability to form hydrophobic interactions with 2′ alanines.

Isolation of a human homolog of osteoclast inhibitory lectin that inhibits the formation and function of osteoclasts.

Osteoclast inhibitory lectin (OCIL) is a newly recognized inhibitor of osteoclast formation. We identified a human homolog of OCIL and its gene, determined its regulation in human osteoblast cell lines, and established that it can inhibit murine and human osteoclast formation and resorption. OCIL shows promise as a new antiresorptive.

Molecular Determinants of Ivermectin Sensitivity at the Glycine Receptor Chloride Channel

Background: The ivermectin-binding site on the glutamate-gated chloride channel was recently resolved by crystallography. Results: Ivermectin binds in a similar orientation to the structurally related glycine receptor, although two H-bonds apparent in the crystal structure proved unimportant for binding to glycine receptors. Conclusion: Ivermectin-binding mechanisms vary among Cys-loop receptors. Significance: Understanding ivermectin-binding mechanisms may help in designing new drugs. Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala288 and Pro230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu218, Ser260, and Thr285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser260 and Thr285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2–M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.