Pharmacological and Biophysical Characteristics of Picrotoxin-Resistant, δSubunit-Containing GABAA Receptors

GABAA receptors (GABAARs) play a crucial role in inhibition in the central nervous system. GABAARs containing the δ subunit mediate tonic inhibition, have distinctive pharmacological properties and are associated with disorders of the nervous system. To explore this receptor sub-class, we recently developed mice with δ-containing receptors rendered resistant to the common non-competitive antagonist picrotoxin (PTX). Resistance was achieved with a knock-in point mutation (T269Y; T6’Y) in the mouse genome. Here we characterize pharmacological and biophysical features of GABAARs containing the mutated subunit to contextualize results from the KI mice. Recombinant receptors containing δ T6’Y plus WT α4 and WT β2 subunits exhibited 3-fold lower EC50 values for GABA but not THIP. GABA EC50 values in native receptors containing the mutated subunit were in the low micromolar range, in contrast with some published results that have suggested nM sensitivity of recombinant receptors. Rectification properties of δ-containing GABAARs were similar to γ2-containing receptors. Receptors containing δ T6’Y had marginally weaker sensitivity to positive allosteric modulators, likely a secondary consequence of differing GABA sensitivity. Overexpression of δT6’Y in neurons resulted in robust PTX-insensitive IPSCs, suggesting that δ-containing receptors are readily recruited by synaptically released GABA. Overall, our results give context to the use of δ receptors with the T6’Y mutation to explore the roles of δ-containing receptors in inhibition.

The Influence of AA29504 on GABAA Receptor Ligand Binding Properties and Its Implications on Subtype Selectivity

The unique pharmacological properties of δ-containing γ-aminobutyric acid type A receptors (δ-GABAARs) make them an attractive target for selective and persistent modulation of neuronal excitability. However, the availability of selective modulators targeting δ-GABAARs remains limited. AA29504 ([2-amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester), an analog of K+ channel opener retigabine, acts as an agonist and a positive allosteric modulator (Ago-PAM) of δ-GABAARs. Based on electrophysiological studies using recombinant receptors, AA29504 was found to be a more potent and effective agonist in δ-GABAARs than in γ2-GABAARs. In comparison, AA29504 positively modulated the activity of recombinant δ-GABAARs more effectively than γ2-GABAARs, with no significant differences in potency. The impact of AA29504's efficacy- and potency-associated GABAAR subtype selectivity on radioligand binding properties remain unexplored. Using [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB) binding assay, we found no difference in the modulatory potency of AA29504 on GABA- and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol)-induced responses between native forebrain GABAARs of wild type and δ knock-out mice. In recombinant receptors expressed in HEK293 cells, AA29504 showed higher efficacy on δ- than γ2-GABAARs in the GABA-independent displacement of [3H]EBOB binding. Interestingly, AA29504 showed a concentration-dependent stimulation of [3H]muscimol binding to γ2-GABAARs, which was absent in δ-GABAARs. This was explained by AA29504 shifting the low-affinity γ2-GABAAR towards a higher affinity desensitized state, thereby rising new sites capable of binding GABAAR agonists with low nanomolar affinity. Hence, the potential of AA29504 to act as a desensitization-modifying allosteric modulator of γ2-GABAARs deserves further investigation for its promising influence on shaping efficacy, duration and plasticity of GABAAR synaptic responses.

5-HT3 receptors in GtoPdb v.2021.3

The 5-HT3 receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on 5-Hydroxytryptamine (serotonin) receptors [69]) is a ligand-gated ion channel of the Cys-loop family that includes the zinc-activated channels, nicotinic acetylcholine, GABAA and strychnine-sensitive glycine receptors. The receptor exists as a pentamer of 4 transmembrane (TM) subunits that form an intrinsic cation selective channel [7]. Five human 5-HT3 receptor subunits have been cloned and homo-oligomeric assemblies of 5-HT3A and hetero-oligomeric assemblies of 5-HT3A and 5-HT3B subunits have been characterised in detail. The 5-HT3C (HTR3C, Q8WXA8), 5-HT3D (HTR3D, Q70Z44) and 5-HT3E (HTR3E, A5X5Y0) subunits [86, 125], like the 5-HT3B subunit, do not form functional homomers, but are reported to assemble with the 5-HT3A subunit to influence its functional expression rather than pharmacological profile [127, 66, 161]. 5-HT3A, -C, -D, and -E subunits also interact with the chaperone RIC-3 which predominantly enhances the surface expression of homomeric 5-HT3A receptor [161]. The co-expression of 5-HT3A and 5-HT3C-E subunits has been demonstrated in human colon [85]. A recombinant hetero-oligomeric 5-HT3AB receptor has been reported to contain two copies of the 5-HT3A subunit and three copies of the 5-HT3B subunit in the order B-B-A-B-A [9], but this is inconsistent with recent reports which show at least one A-A interface [99, 154]. The 5-HT3B subunit imparts distinctive biophysical properties upon hetero-oligomeric 5-HT3AB versus homo-oligomeric 5-HT3A recombinant receptors [35, 44, 59, 88, 143, 132, 82], influences the potency of channel blockers, but generally has only a modest effect upon the apparent affinity of agonists, or the affinity of antagonists ([19], but see [44, 33, 38]) which may be explained by the orthosteric binding site residing at an interface formed between 5-HT3A subunits [99, 154]. However, 5-HT3A and 5-HT3AB receptors differ in their allosteric regulation by some general anaesthetic agents, small alcohols and indoles [142, 139, 73]. The potential diversity of 5-HT3 receptors is increased by alternative splicing of the genes HTR3A and HTR3E [67, 21, 127, 126, 123]. In addition, the use of tissue-specific promoters driving expression from different transcriptional start sites has been reported for the HTR3A, HTR3B, HTR3D and HTR3E genes, which could result in 5-HT3 subunits harbouring different N-termini [156, 82, 123]. To date, inclusion of the 5-HT3A subunit appears imperative for 5-HT3 receptor function.

New Human Follitropin Preparations: How Glycan Structural Differences May Affect Biochemical and Biological Function and Clinical Effect

It is well accepted that pituitary follitropin is secreted into the circulation as a mixture of variants, which differ not in primary structure but rather at the level of glycosylation. These glycosidic forms vary in the number of glycosylation sites filled, complexity of glycosidic chains, and sialylation and sulfation. It is generally agreed that high sialylation, 2,3 sialic acid capping of terminal N-acetyl galactosamine or galactose leads to longer circulating half-life, by blocking binding of asialoglycoprotein receptor (ASGPR) in the liver. In contrast, 2,6 sialic acid found in humans does not prevent recognition of galactose and N-acetyl galactosamine by ASGPR. Few studies on clinical outcomes comparing differences in sialylation of follitropin found in commercially available preparations are available. Thus, there is a clear need for a consortium of open data to address this unmet need. Recently, FSH glycosylation, primarily on the β-subunit, which varies as women age, has emerged as a key modifier of follitropin action, with profound biological effects in vivo in animal models. To date, limited information of recombinant follitropin hormone preparations is available. Thus, most of the studies with FSH that is well characterized biochemically have been done in vitro, with engineered non gonadal host cells bearing recombinant receptors or in animal models. Since limited studies in human granulosa cells are available, a question is whether structural differences in glycosylation in commercially available follitropin affects biological function and clinical effect in humans. The presence of fucose, for example, has not been studied greatly even though, in the case of antibody therapy it has been shown to have a large effect on antibody targeting. This review on glycosidic variability of follitropin from the biochemical/structural point of view reflects on this question and presents an assessment in the context of available published data. If clinical differences are to be expected or not, the readers will have a better understanding of the evidence for and limitations of such expectations.

AMPAR/TARP stoichiometry differentially modulates channel properties

AMPARs control fast synaptic communication between neurons and their function relies on auxiliary subunits, which importantly modulate channel properties. Although it has been suggested that AMPARs can bind to TARPs with variable stoichiometry, little is known about the effect that this stoichiometry exerts on certain AMPAR properties. Here we have found that AMPARs show a clear stoichiometry-dependent modulation by the prototypical TARP γ2 although the receptor still needs to be fully saturated with γ2 to show some typical TARP-induced characteristics (i.e. an increase in channel conductance). We also uncovered important differences in the stoichiometric modulation between calcium-permeable and calcium-impermeable AMPARs. Moreover, in heteromeric AMPARs, γ2 positioning in the complex is important to exert certain TARP-dependent features. Finally, by comparing data from recombinant receptors with endogenous AMPAR currents from mouse cerebellar granule cells, we have determined a likely presence of two γ2 molecules at somatic receptors in this cell type.

AMPAR/TARP stoichiometry differentially modulates channel properties

SummaryAMPARs control fast synaptic communication between neurons and their function relies on auxiliary subunits, which importantly modulate channel properties. Although it has been suggested that AMPARs can bind to TARPs with variable stoichiometry, little is known about the effect that this stoichiometry exerts on certain AMPAR properties. Here we have found that AMPARs show a clear stoichiometry dependent modulation although AMPARs still need to be fully saturated with TARPs to show some typical TARP-induced characteristics (i.e. an increase in channel conductance). We also have uncovered important differences in the stoichiometric modulation between calcium-permeable and calcium-impermeable AMPARs. Moreover, in heteromeric AMPARs, TARP positioning in the complex is important to exert certain TARP-dependent features. Finally, by comparing data from recombinant receptors with endogenous AMPAR currents from cerebellar granule cells, we have determined a likely functional stoichiometry of 2 TARPs associated with GluA2 subunits in the somatic AMPARs found in this cell type.

Mild chronic perturbation of inhibition severely alters hippocampal function

Pentameric GABAA receptors mediate a large share of CNS inhibition. The γ2 subunit is a typical constituent. At least 11 mutations in the γ2 subunit cause human epilepsies, making the role of γ2-containing receptors in brain function of keen basic and translational interest. How small changes to inhibition may cause brain abnormalities, including seizure disorders, is unclear. In mice, we perturbed fast inhibition with a point mutation T272Y (T6′Y in the second membrane-spanning domain) to the γ2 subunit. The mutation imparts resistance to the GABAA receptor antagonist picrotoxin, allowing verification of mutant subunit incorporation. We confirmed picrotoxin resistance and biophysical properties in recombinant receptors. T6′Y γ2-containing receptors also exhibited faster deactivation but unaltered steady-state properties. Adult T6′Y knockin mice exhibited myoclonic seizures and abnormal cortical EEG, including abnormal hippocampal-associated theta oscillations. In hippocampal slices, picrotoxin-insensitive inhibitory synaptic currents exhibited fast decay. Excitatory/inhibitory balance was elevated by an amount expected from the IPSC alteration. Partial pharmacological correction of γ2-mediated IPSCs with diazepam restored total EEG power toward baseline, but had little effect on the abnormal low-frequency peak in the EEG. The results suggest that at least part of the abnormality in brain function arises from the acute effects of truncated inhibition.

5-HT3 receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The 5-HT3 receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on 5-Hydroxytryptamine (serotonin) receptors [66]) is a ligand-gated ion channel of the Cys-loop family that includes the zinc-activated channels, nicotinic acetylcholine, GABAA and strychnine-sensitive glycine receptors. The receptor exists as a pentamer of 4TM subunits that form an intrinsic cation selective channel [5]. Five human 5-HT3 receptor subunits have been cloned and homo-oligomeric assemblies of 5-HT3A and hetero-oligomeric assemblies of 5-HT3A and 5-HT3B subunits have been characterised in detail. The 5-HT3C (HTR3C, Q8WXA8), 5-HT3D (HTR3D, Q70Z44) and 5-HT3E (HTR3E, A5X5Y0) subunits [83, 122], like the 5-HT3B subunit, do not form functional homomers, but are reported to assemble with the 5-HT3A subunit to influence its functional expression rather than pharmacological profile [124, 63, 157]. 5-HT3A, -C, -D, and -E subunits also interact with the chaperone RIC-3 which predominantly enhances the surface expression of homomeric 5-HT3A receptor [157]. The co-expression of 5-HT3A and 5-HT3C-E subunits has been demonstrated in human colon [82]. A recombinant hetero-oligomeric 5-HT3AB receptor has been reported to contain two copies of the 5-HT3A subunit and three copies of the 5-HT3B subunit in the order B-B-A-B-A [7], but this is inconsistent with recent reports which show at least one A-A interface [96, 150]. The 5-HT3B subunit imparts distinctive biophysical properties upon hetero-oligomeric 5-HT3AB versus homo-oligomeric 5-HT3A recombinant receptors [32, 41, 56, 85, 139, 129, 79], influences the potency of channel blockers, but generally has only a modest effect upon the apparent affinity of agonists, or the affinity of antagonists ([17], but see [41, 30, 35]) which may be explained by the orthosteric binding site residing at an interface formed between 5-HT3A subunits [96, 150]. However, 5-HT3A and 5-HT3AB receptors differ in their allosteric regulation by some general anaesthetic agents, small alcohols and indoles [138, 135, 71]. The potential diversity of 5-HT3 receptors is increased by alternative splicing of the genes HTR3A and E [64, 19, 124, 123, 120]. In addition, the use of tissue-specific promoters driving expression from different transcriptional start sites has been reported for the HTR3A, HTR3B, HTR3D and HTR3E genes, which could result in 5-HT3 subunits harbouring different N-termini [152, 79, 120]. To date, inclusion of the 5-HT3A subunit appears imperative for 5-HT3 receptor function.

From T-cell Engineering to CAR therapy: Progress and Prospects

The genetic engineering of T cells provides a means to rapidly generate anti-tumor T cells for any cancer patient. This approach is predicated on gene transfer technology that enables the expression of receptors for antigen and other gene products in primary T cells. Tumor targeting may be achieved through the transfer of a physiological receptor for antigen, which is known as the T cell receptor (TCR), or synthetic fusion receptors, which we grouped under the general term of chimeric antigen receptor (CAR). CARs are recombinant receptors for antigen, which, in a single molecule, redirect T cell specificity and eventually enhance anti-tumor potency. Functional augmentation is achieved through the design of second generation CARs, which not only redirect cytotoxicity, but also reprogram T cell function and longevity through their costimulatory properties. The combined activating and costimulatory domains incorporated in second-generation CARs critically determine the function, differentiation, metabolism and persistence of engineered T cells. CD19 CARs that incorporate CD28 or 4-1BB signalling domains are the best known to date. Two decades ago, we selected CD19 as the prime target for developing our CAR technology and provided the first proof-of-principle that CD19-targeted human peripheral blood T cells could eradicate a broad range of B cell malignancies in immunodeficient mice (Brentjens RJ, Riviere I, et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. 2003;9(3):279-86). CD19 has since become the poster child for CAR therapies. Complete remissions have been reported from several centers in patients with non-Hodgkin lymphoma, chronic lymphocytic leukemia and, most dramatically, acute lymphoblastic leukemia. Two types of second generation CARs, utilizing either CD28 or 4-1BB as their costimulatory signaling components, have been used in ALL patients. Both have yielded dramatic outcomes, in adults as well as in children. Our data indicate that CD28-based CARs direct a brisk proliferative response and boost effector functions, while 4-1BB-based CARs direct a gradual T cell accumulation that may eventually overcome lesser functional potency. These distinct kinetic features can be exploited to further develop CAR T cell therapies for a variety of cancers. We have now modeled CD19 CAR therapy for ALL in a "stress test", wherein we purposefully lower the infused T cell doses to challenge the CAR therapy. We have compared novel CAR designs intended to recruit both CD28 and 4-1BB signaling. These quantitative analyses reveal striking disparities that hinge on subtle variations in the structural design of CARs and co-expressed costimulatory molecules. Remarkably, we find that some of the most effective engineering strategies activate and sustain the recruitment of the IFNβ pathway through the induction of IRF7, while lowering the induction of exhaustion markers relative to second generation CARs activating either CD28 or 4-1BB alone. The field is thus poised to move beyond the CD28 vs 4-1BB debate, which will be rendered obsolete by the emergence of superior CAR designs that coopt the use of costimulatory ligands, cytokines and/or checkpoint blockade inhibitors. A new field of immunopharmacology is emerging. Disclosures Sadelain: Juno Therapeutics: Consultancy, Equity Ownership, Other: Co-Founder, stockholder, Patents & Royalties: Licensed patents on CARs.