Conformational plasticity of human acid-sensing ion channel 1a.

Acid-sensing ion channels (ASICs) are typically activated by acidic environments and contribute to nociception and synaptic plasticity. ASIC1a is the most abundant subunit in the central nervous system and forms homomeric channels permeable to Na and Ca , making it a compelling therapeutic target for acidotic pathologies including stroke and traumatic brain injury. However, a complete conformational library of human ASIC1a in its various functional states has yet to be described.

P2X2 receptor subunit interfaces are missense variant hotspots, where mutations tend to increase apparent ATP affinity.

Background And Purpose: P2X receptors are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains.

Crosslinking glutamate receptor ion channels.

Combining crosslinking strategies with electrophysiology, biochemistry, and structural in silico analysis is a powerful tool to study transient movements of ion channels during gating. This chapter describes crosslinking in living cells using cysteine and photoactive unnatural amino acids (UAAs) that we have used on glutamate receptor ion channels. Here, we share the protocol for building a perfusion tool to enable rapid chemical modification of glutamate-gated AMPA receptors, optimized for their fast activation.

Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis.

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the vertebrate nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type iGluRs using genetically encoded unnatural amino acid (UAA) photocross-linkers, p-benzoyl-l-phenylalanine (BzF) and p-azido-l-phenylalanine (AzF).

Controlling Ca Permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors with Photochromic Ion Channel Blockers.

Ionotropic glutamate receptors (iGluRs) play a critical role in normal brain function and neurodegenerative diseases. Development of light-dependent compounds would enable studies of iGluRs within intact mammalian neural tissue, as light is noninvasive and can be applied with high spatiotemporal precision. Here we develop a potent photochromic antagonist that selectively targets the Ca permeable AMPA-type of iGuRs, thus providing an important tool to study the contribution of AMPA-type iGluRs on neuronal activity.

Structure-activity relationship study of spider polyamine toxins as inhibitors of ionotropic glutamate receptors.

The spider polyamine toxins Joro spider toxin-3 (JSTX-3) and Nephila polyamine toxins-1 and -8 (NPTX-1 and NPTX-8) are isolated from the venom of the orb-weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open-channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid-phase synthetic methodology.

Structure-activity relationship studies of N-methylated and N-hydroxylated spider polyamine toxins as inhibitors of ionotropic glutamate receptors.

Polyamine toxins from spiders and wasps are potent open-channel blockers of ionotropic glutamate (iGlu) receptors. It is well-established that secondary amino groups in the polyamine moiety of these toxins are key to both selectivity and potency at iGlu receptors, still some native spider polyamine toxins comprise both N-methyl and N-hydroxy functionalities. Here, we investigate the effect of both N-methylation and N-hydroxylation of spider polyamine toxins by the synthesis and biological evaluation of the naturally occurring N-methylated argiopinines and pseudoargiopinines I and II, N-hydroxylated Agel-489 and Agel-505, as well as N-methylated analogues of the NMDA and AMPA iGlu receptor subtype selective antagonists ArgTX-93 and ArgTX-48.

Inhibition of AMPA receptors by polyamine toxins is regulated by agonist efficacy and stargazin.

The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are glutamate-gated cation channels mediating the majority of fast excitatory synaptic transmission in the central nervous system (CNS). Polyamine toxins derived from spiders and wasps are use- and voltage-dependent channel blockers of Ca(2+)-permeable AMPARs. Recent studies have suggested that AMPAR block by polyamine toxins is modulated by auxiliary subunits from the class of transmembrane AMPAR regulatory proteins (TARPs), which may have implications for their use as tool compounds in native systems.

Evaluation of PhTX-74 as subtype-selective inhibitor of GluA2-containing AMPA receptors.

The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are glutamate-gated cation channels that mediate fast excitatory synaptic transmission in the central nervous system. AMPARs are tetramers formed by homo- or heteromeric assembly of GluA1-4 subunits to produce multiple subtypes with varying biophysical properties. Polyamine toxins such as joro spider toxins, philanthotoxins (PhTXs), and argiotoxins are use-dependent ion channel blockers of AMPARs widely employed as highly potent antagonists of GluA2-lacking receptor subtypes.

Structure-activity relationship studies of argiotoxins: selective and potent inhibitors of ionotropic glutamate receptors.

Argiotoxin-636 (ArgTX-636), a natural product from the spider Argiope lobata, is a potent but nonselective open-channel blocker of ionotropic glutamate (iGlu) receptors. Here, three series of analogues were designed to exploit selectivity among iGlu receptors, taking advantage of a recently developed solid-phase synthetic methodology for the synthesis of ArgTX-636 and analogues. Initially, the importance of secondary amino groups in the polyamine chain was studied by the synthesis of systematically modified ArgTX-636 analogues, which were evaluated for pharmacological activity at NMDA and AMPA receptors.

General synthesis of β-alanine-containing spider polyamine toxins and discovery of nephila polyamine toxins 1 and 8 as highly potent inhibitors of ionotropic glutamate receptors.

Certain spiders contain large pools of polyamine toxins, which are putative pharmacological tools awaiting further discovery. Here we present a general synthesis strategy for this class of toxins and prepare five structurally varied polyamine toxins. Electrophysiological testing at three ionotropic glutamate receptor subtypes reveals that two of these, Nephila polyamine toxins 1 (NPTX-1) and 8 (NPTX-8), comprise intriguing pharmacological activities by having subnanomolar IC(50) values at kainate receptors.

Solid-phase synthesis and biological evaluation of Joro spider toxin-4 from Nephila clavata.

Polyamine toxins from orb weaver spiders are attractive pharmacological tools particularly for studies of ionotropic glutamate (iGlu) receptors in the brain. These polyamine toxins are biosynthesized in a combinatorial manner, providing a plethora of related, but structurally complex toxins to be exploited in biological studies. Here, we have used solid-phase synthetic methodology for the efficient synthesis of Joro spider toxin-4 (JSTX-4) (1) from Nephila clavata, providing sufficient amounts of the toxin for biological evaluation at iGlu receptor subtypes using electrophysiology.