Tricyclic antipsychotics and antidepressants can inhibit α5-containing GABAA receptors by two distinct mechanisms

Background and Purpose: Many psychotherapeutic drugs, including clozapine, display polypharmacology and act on GABA receptors. Patients with schizophrenia show alterations in function, structure and molecular composition of the hippocampus, and a recent study demonstrated aberrant levels of hippocampal a5 subunit-containing GABA receptors. The purpose of this study is to investigate tricyclic compounds in a5 subunit-containing receptor subtypes. Experimental Approach: Functional studies of effects by seven antipsychotic and antidepressant medications were performed in several GABA receptor subtypes by two‐electrode voltage‐clamp electrophysiology using Xenopus laevis oocytes. Computational structural analysis was employed to design mutated constructs of the a5 subunit, probing a novel binding site. Radioligand displacement data complemented the functional and mutational findings. Key Results: We show that the antipsychotic drugs clozapine and chlorpromazine exert functional inhibition on multiple GABA receptor subtypes, including a5-containing ones. Based on a chlorpromazine binding site observed in a GABA-gated bacterial homologue, we identified a novel site in a5 GABA receptor subunits and demonstrate differential usage of this and the orthosteric sites by these ligands. Conclusion and Implications: Despite high molecular and functional similarities among the tested ligands, they reduce GABA currents by differential usage of allosteric and orthosteric sites. The CPZ site we describe here is a new potential target for optimizing antipsychotic medications with beneficial polypharmacology. Further studies in defined subtypes are needed to substantiate mechanistic links between the therapeutic effects of clozapine and its action on certain GABA receptor subtypes.

Tricyclic antipsychotics and antidepressants can inhibit α5-containing GABAA receptors by two distinct mechanisms

Background and Purpose: Many psychotherapeutic drugs, including clozapine, display polypharmacology and act on GABA receptors. Patients with schizophrenia show alterations in function, structure and molecular composition of the hippocampus, and a recent study demonstrated aberrant levels of hippocampal a5 subunit-containing GABA receptors. The purpose of this study is to investigate tricyclic compounds in a5 subunit-containing receptor subtypes. Experimental Approach: Functional studies of effects by seven antipsychotic and antidepressant medications were performed in several GABA receptor subtypes by two‐electrode voltage‐clamp electrophysiology using Xenopus laevis oocytes. Computational structural analysis was employed to design mutated constructs of the a5 subunit, probing a novel binding site. Radioligand displacement data complemented the functional and mutational findings. Key Results: We show that the antipsychotic drugs clozapine and chlorpromazine exert functional inhibition on multiple GABA receptor subtypes, including a5-containing ones. Based on a chlorpromazine binding site observed in a GABA-gated bacterial homologue, we identified a novel site in a5 GABA receptor subunits and demonstrate differential usage of this and the orthosteric sites by these ligands. Conclusion and Implications: Despite high molecular and functional similarities among the tested ligands, they reduce GABA currents by differential usage of allosteric and orthosteric sites. The C C C C C C site we describe here is a new potential target for optimizing antipsychotic medications with beneficial polypharmacology. Further studies in defined subtypes are needed to substantiate mechanistic links between the therapeutic effects of clozapine and its action on certain GABA receptor subtypes.

The Caenorhabditis elegans DEG-3/DES-2 Channel Is a Betaine-Gated Receptor Insensitive to Monepantel

Natural plant compounds, such as betaine, are described to have nematocidal properties. Betaine also acts as a neurotransmitter in the free-living model nematode Caenorhabditis elegans, where it is required for normal motility. Worm motility is mediated by nicotinic acetylcholine receptors (nAChRs), including subunits from the nematode-specific DEG-3 group. Not all types of nAChRs in this group are associated with motility, and one of these is the DEG-3/DES-2 channel from C. elegans, which is involved in nociception and possibly chemotaxis. Interestingly, the activity of DEG-3/DES-2 channel from the parasitic nematode of ruminants, Haemonchus contortus, is modulated by monepantel and its sulfone metabolite, which belong to the amino-acetonitrile derivative anthelmintic drug class. Here, our aim was to advance the pharmacological knowledge of the DEG-3/DES-2 channel from C. elegans by functionally expressing the DEG-3/DES-2 channel in Xenopus laevis oocytes and using two-electrode voltage-clamp electrophysiology. We found that the DEG-3/DES-2 channel was more sensitive to betaine than ACh and choline, but insensitive to monepantel and monepantel sulfone when used as direct agonists and as allosteric modulators in co-application with betaine. These findings provide important insight into the pharmacology of DEG-3/DES-2 from C. elegans and highlight the pharmacological differences between non-parasitic and parasitic nematode species.

Lack of Charge Interaction in the Ion Binding Site Determines Anion Selectivity in the Sodium Bicarbonate Cotransporter NBCe1

The Na/HCO3 cotransporter NBCe1 is a member of SLC4A transporters that move HCO3− across cell membranes and regulate intracellular pH or transepithelial HCO3 transport. NBCe1 is highly selective to HCO3− and does not transport other anions; the molecular mechanism of anion selectivity is presently unclear. We previously reported that replacing Asp555 with a Glu (D555E) in NBCe1 induces increased selectivity to other anions, including Cl−. This finding is unexpected because all SLC4A transporters contain either Asp or Glu at the corresponding position and maintain a high selectivity to HCO3−. In this study, we tested whether the Cl− transport in D555E is mediated by an interaction between residues in the ion binding site. Human NBCe1 and mutant transporters were expressed in Xenopus oocytes, and their ability to transport Cl− was assessed by two-electrode voltage clamp. The results show that the Cl− transport is induced by a charge interaction between Glu555 and Lys558. The bond length between the two residues is within the distance for a salt bridge, and the ionic strength experiments confirm an interaction. This finding indicates that the HCO3− selectivity in NBCe1 is established by avoiding a specific charge interaction in the ion binding site, rather than maintaining such an interaction.

TACAN is a novel regulator of PKD2

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in membrane receptor PKD1 or cation channel PKD2. TACAN (also named TMEM120A), recently reported as an ion channel in neuron cells for mechano and pain sensing, is also distributed in diverse non-neuronal tissues such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes we found that TACAN inhibited the channel activity of PKD2 gain-of-function mutant F604P. The first and last transmembrane domains of TACAN were found to interact with the PKD2 C- and N-terminal portions, respectively. We showed that the TACAN N-terminus acted as a blocking peptide and that TACAN inhibits the PKD2 function through the PKD2/TACAN binding. By patch clamping in mammalian cells, we found that TACAN inhibits both the single channel conductance and open probability of PKD2 and mutant F604P. Further, PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechano sensitivity of the PKD2/TACAN channel complex.

Interaction of α9α10 Nicotinic Receptors With Peptides and Proteins From Animal Venoms

Unlike most neuronal nicotinic acetylcholine receptor (nAChR) subunits, α7, α9, and α10 subunits are able to form functional homo- or heteromeric receptors without any β subunits. While the α7 subtype is widely distributed in the mammalian brain and several peripheral tissues, α9 and α9α10 nAChRs are mainly found in the cochlea and immune cells. α-Conotoxins that specifically block the α9α10 receptor showed anti-nociceptive and anti-hyperalgesic effects in animal models. Hence, this subtype is considered a drug target for analgesics. In contrast to the α9α10-selective α-conotoxins, the three-finger toxin α-bungarotoxin inhibits muscle-type and α7 nAChRs in addition to α9α10 nAChRs. However, the selectivity of α-neurotoxins at the α9α10 subtype was less intensively investigated. Here, we compared the potencies of α-conotoxins and α-neurotoxins at the human α9α10 nAChR by two-electrode voltage clamp analysis upon expression in Xenopus oocytes. In addition, we analyzed effects of several α9α10-selective α-conotoxins on mouse granulocytes from bone marrow to identify possible physiological functions of the α9α10 nAChR subtype in these cells. The α-conotoxin-induced IL-10 release was measured upon LPS-stimulation. We found that α-conotoxins RgIA, PeIA, and Vc1.1 enhance the IL-10 expression in granulocytes which might explain the known anti-inflammatory and associated analgesic activities of α9α10-selective α-conotoxins. Furthermore, we show that two long-chain α-neurotoxins from the cobra Naja melanoleuca venom that were earlier shown to bind to muscle-type and α7 nAChRs, also inhibit the α9α10 subtype at nanomolar concentrations with one of them showing a significantly slower dissociation from this receptor than α-bungarotoxin.

Activation of SGK1.1 Upregulates the M-current in the Presence of Epilepsy Mutations

In the central nervous system, the M-current plays a critical role in regulating subthreshold electrical excitability of neurons, determining their firing properties and responsiveness to synaptic input. The M-channel is mainly formed by subunits Kv7.2 and Kv7.3 that co-assemble to form a heterotetrametric channel. Mutations in Kv7.2 and Kv7.3 are associated with hyperexcitability phenotypes including benign familial neonatal epilepsy (BFNE) and neonatal epileptic encephalopathy (NEE). SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), increases M-current density in neurons, leading to reduced excitability and protection against seizures. Herein, using two-electrode voltage clamp on Xenopus laevis oocytes, we demonstrate that SGK1.1 selectively activates heteromeric Kv7 subunit combinations underlying the M-current. Importantly, activated SGK1.1 increases M-channel activity in the presence of two different epilepsy mutations found in Kv7.2, R207W and A306T. In addition, proximity ligation assays in the N2a cell line allowed us to address the effect of these mutations on Kv7-SGK1.1-Nedd4 molecular associations, a proposed pathway underlying augmentation of M-channel activity by SGK1.1

Spatiotemporal control of femtosecond laser filament-triggered discharge and its application in diagnosing gas flow fields

Precise control of the discharge in space and time is of great significance for better applications of discharge plasma. Here, we used a femtosecond laser filament to trigger and guide a high-voltage DC pulse discharge to achieve spatiotemporal control of the discharge plasma. In space, the discharge plasma is distributed strictly along the channel generated by the femtosecond laser filament. The breakdown voltage threshold is reduced, and the discharge length is extended. In time, the electrical parameters such as the electrode voltage and the electrode gap affect discharge delay time and jitter. By optimizing the parameters, we can achieve sub-nanosecond jitter of the discharge. Based on the spatiotemporal control of the discharge, we applied filament-triggered discharge for one-dimensional composition measurements of the gas flow field. Besides, the technique shows great potential in studying the spatiotemporal evolution of discharge plasma.

Activation of SGK1.1 up-regulates the M-current in the presence of epilepsy mutations

In the central nervous system, the M-current plays a critical role in regulating subthreshold electrical excitability of neurons, determining their firing properties and responsiveness to synaptic input. The M-channel is mainly formed by subunits Kv7.2 and Kv7.3 that co-assemble to form a heterotetrametric channel. Mutations in Kv7.2 and Kv7.3 are associated with hyperexcitability phenotypes including benign familial neonatal epilepsy (BFNE) and neonatal epileptic encephalopathy (NEE). SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), increases M-current density in neurons, leading to reduced excitability and protection against seizures. Herein, using two-electrode voltage clamp on Xenopus laevis oocytes, we demonstrate that SGK1.1 selectively activates heteromeric Kv7 subunit combinations underlying the M-current. Importantly, activated SGK1.1 is able to up-regulate M-channel activity in the presence of two different epilepsy mutations found in Kv7.2 subunit, R207W and A306T. In addition, proximity ligation assays in the N2a cell line allowed us to address the effect of these mutations on Kv7-SGK1.1-Nedd4 molecular associations, a proposed pathway underlying M-channel up-regulation by SGK1.1

A novel ion conducting route besides the central pore in an inherited mutant of G-protein-gated inwardly rectifying K+ channel

G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important physiological roles in various organs. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited GIRK mutants. By the two-electrode voltage-clamp analysis of GIRK mutants heterologously expressed in Xenopus oocytes, we observed that Kir3.2 G156S permeates Li+ better than Rb+, while T154del or L173R of Kir3.2 and T158A of Kir3.4 permeate Rb+ better than Li+, suggesting a unique conformational change in the G156S mutant. Applications of blockers of the selectivity filter (SF) pathway, Ba2+ or Tertiapin-Q (TPN-Q), remarkably increased the Li+-selectivity of Kir3.2 G156S but did not alter those of the other mutants. In single-channel recordings of Kir3.2 G156S expressed in mouse fibroblasts, two types of events were observed, one attributable to a TPN-Q sensitive K+ current and the second a TPN-Q resistant Li+ current. The results show that a novel Li+ permeable and blocker-resistant pathway exists in G156S in addition to the SF pathway. Mutations in the pore helix (PH), S148F and T151A, also induced high Li+ permeation. Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations.