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"Miller, Keith"
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Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer
Type A γ-aminobutyric acid (GABA
A
) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system
1
,
2
. Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia
3
,
4
. Among the numerous assemblies that are theoretically possible, the most prevalent in the brain are the α1β2/3γ2 GABA
A
receptors
5
. The β3 subunit has an important role in maintaining inhibitory tone, and the expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in β1–β3 triple knockout neurons
6
. So far, efforts to generate accurate structural models for heteromeric GABA
A
receptors have been hampered by the use of engineered receptors and the presence of detergents
7
–
9
. Notably, some recent cryo-electron microscopy reconstructions have reported ‘collapsed’ conformations
8
,
9
; however, these disagree with the structure of the prototypical pentameric ligand-gated ion channel the
Torpedo
nicotinic acetylcholine receptor
10
,
11
, the large body of structural work on homologous homopentameric receptor variants
12
and the logic of an ion-channel architecture. Here we present a high-resolution cryo-electron microscopy structure of the full-length human α1β3γ2L—a major synaptic GABA
A
receptor isoform—that is functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator ‘megabody’ and is in a desensitized conformation. Each GABA
A
receptor pentamer contains two phosphatidylinositol-4,5-bisphosphate molecules, the head groups of which occupy positively charged pockets in the intracellular juxtamembrane regions of α1 subunits. Beyond this level, the intracellular M3–M4 loops are largely disordered, possibly because interacting post-synaptic proteins are not present. This structure illustrates the molecular principles of heteromeric GABA
A
receptor organization and provides a reference framework for future mechanistic investigations of GABAergic signalling and pharmacology.
A high-resolution cryo-electron microscopy structure is reported for the full-length human α1β3γ2L GABA
A
receptor, functionally reconstituted in lipid nanodiscs.
Journal Article
GABAA receptor signalling mechanisms revealed by structural pharmacology
Type-A γ-aminobutyric (GABA
A
) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABA
A
receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABA
A
receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABA
A
receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABA
A
receptor modulators.
Cryo-electron microscopy structures are reported in which the full-length human α1β3γ2L GABA
A
receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA, and the benzodiazepines alprazolam and diazepam.
Journal Article
Hypoalbuminemia and Clinical Outcomes: What is the Mechanism behind the Relationship?
Albumin has a number of important physiologic functions, which include maintaining oncotic pressure, transporting various agents (fatty acids, bile acids, cholesterol, metal ions, and drugs), scavenging free oxygen radicals, acting as an antioxidant, and exerting an antiplatelet effect. Hypoalbuminemia in adults, defined by an intravascular albumin level of <3.5 g/dL, is associated with poor postoperative outcomes in patients undergoing surgical intervention. Although the relationship of hypoalbuminemia and poor surgical outcome has been known for many years, the pathophysiology behind the relationship is unclear. Three theoretical constructs might explain this relationship. First, albumin might serve as a nutritional marker, such that hypoalbuminemia represents poor nutritional status in patients who go on to experience poor postoperative outcomes. Second, albumin has its own pharmacologic characteristics as an antioxidant or transporter, and therefore, the lack of albumin might result in a deficiency of those functions, resulting in poor postoperative outcomes. Or third, albumin is known to be a negative acute phase protein, and as such hypoalbuminemia might represent an increased inflammatory status of the patient, potentially leading to poor outcomes. A thorough review of the literature reveals the fallacy of these arguments and fails to show a direct cause and effect between low albumin levels per se and adverse outcomes. Interventions designed solely to correct preoperative hypoalbuminemia, in particular intravenous albumin infusion, do little to change the patient's course of hospitalization. While surgeons may use albumin levels on admission for their prognostic value, they should avoid therapeutic strategies whose main endpoint is correction of this abnormality.
Journal Article
GABA A receptor signalling mechanisms revealed by structural pharmacology
Type-A γ-aminobutyric (GABA
) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABA
receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABA
receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABA
receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABA
receptor modulators.
Journal Article
Differential assembly diversifies GABAA receptor structures and signalling
Type A γ-aminobutyric acid receptors (GABA
A
Rs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits
1
,
2
and can be modulated by essential medicines including general anaesthetics and benzodiazepines
3
. Human GABA
A
R subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABA
A
Rs assembled from α4, β3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors
4
–
7
. This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABA
A
R arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data
8
,
9
, we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABA
A
Rs.
The diverse makeup and assembly of subunits augment the structure, physiology and pharmacology of GABA
A
receptors.
Journal Article
Self-Driving Cars and Engineering Ethics: The Need for a System Level Analysis
The literature on self-driving cars and ethics continues to grow. Yet much of it focuses on ethical complexities emerging from an individual vehicle. That is an important but insufficient step towards determining how the technology will impact human lives and society more generally. What must complement ongoing discussions is a broader, system level of analysis that engages with the interactions and effects that these cars will have on one another and on the socio-technical systems in which they are embedded. To bring the conversation of self-driving cars to the system level, we make use of two traffic scenarios which highlight some of the complexities that designers, policymakers, and others should consider related to the technology. We then describe three approaches that could be used to address such complexities and their associated shortcomings. We conclude by bringing attention to the “Moral Responsibility for Computing Artifacts: The Rules”, a framework that can provide insight into how to approach ethical issues related to self-driving cars.
Journal Article
Novel Anti-Nicotine Vaccine Using a Trimeric Coiled-Coil Hapten Carrier
Tobacco addiction represents one of the largest public health problems in the world and is the leading cause of cancer and heart disease, resulting in millions of deaths a year. Vaccines for smoking cessation have shown considerable promise in preclinical models, although functional antibody responses induced in humans are only modestly effective in preventing nicotine entry into the brain. The challenge in generating serum antibodies with a large nicotine binding capacity is made difficult by the fact that this drug is non-immunogenic and must be conjugated as a hapten to a protein carrier. To circumvent the limitations of traditional carriers like keyhole limpet hemocyanin (KLH), we have synthesized a short trimeric coiled-coil peptide (TCC) that creates a series of B and T cell epitopes with uniform stoichiometry and high density. Here we compared the relative activities of a TCC-nic vaccine and two control KLH-nic vaccines using Alum as an adjuvant or GLA-SE, which contains a synthetic TLR4 agonist formulated in a stable oil-in-water emulsion. The results showed that the TCC's high hapten density correlated with a better immune response in mice as measured by anti-nicotine Ab titer, affinity, and specificity, and was responsible for a reduction in anti-carrier immunogenicity. The Ab responses achieved with this synthetic vaccine resulted in a nicotine binding capacity in serum that could prevent >90% of a nicotine dose equivalent to three smoked cigarettes (0.05 mg/kg) from reaching the brain.
Journal Article
T Cell Receptor-Like Recognition of Tumor In Vivo by Synthetic Antibody Fragment
A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR) binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC) molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab) library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu) peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2), with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64)Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT) imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.
Journal Article