Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is up-regulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs, and noncanonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is primarily mediated through electrostatic interactions of basic amino acids in the BigDyn N terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the noncanonical amino acid azidophenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn, and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.

https://www.pnas.org/content/pnas/117/13/7447.full.pdf

Mechanism and site of action of big dynorphin on ASIC1a.

Intercellular signaling events mediated by neuropeptides and peptide hormones represent important targets for both basic science and drug discovery For many bioactive peptides, the protein receptors that transmit information across the receiving cell membrane are not known, severely limiting these signaling pathways as potential therapeutic targets Identifying the receptor(s) for a given peptide of interest is complicated by several factors Most notably, cell-cell signaling peptides are generated through dynamic biosynthetic pathways, can act on many different families of receptor proteins, and can participate in complex ligand-receptor interactions that extend beyond a simple one-to-one archetype Here, we discuss recent methodological advances to identify signaling partners for bioactive peptides Recent efforts have centered on methods to identify candidate receptors via transcript expression, methods to match peptide-receptor pairs through high throughput screening, and methods to capture direct ligand-receptor interactions using chemical probes Future applications of the receptor identification approaches discussed here, as well as technical advancements to address their limitations, promise to lead to a greater understanding of how cells communicate to deliver complex physiologies Importantly, such advancements will likely provide novel targets for the treatment of human diseases within the central nervous and endocrine systems

Identifying Receptors for Neuropeptides and Peptide Hormones: Challenges and Recent Progress.

Abstract Pain is estimated to affect more than 20% of the global population, imposing incalculable health and economic burdens. Effective pain management is crucial for individuals suffering from pain. However, the current methods for pain assessment and treatment fall short of clinical needs. Benefiting from advances in neuroscience and biotechnology, the neuronal circuits and molecular mechanisms critically involved in pain modulation have been elucidated. These research achievements have incited progress in identifying new diagnostic and therapeutic targets. In this review, we first introduce fundamental knowledge about pain, setting the stage for the subsequent contents. The review next delves into the molecular mechanisms underlying pain disorders, including gene mutation, epigenetic modification, posttranslational modification, inflammasome, signaling pathways and microbiota. To better present a comprehensive view of pain research, two prominent issues, sexual dimorphism and pain comorbidities, are discussed in detail based on current findings. The status quo of pain evaluation and manipulation is summarized. A series of improved and innovative pain management strategies, such as gene therapy, monoclonal antibody, brain-computer interface and microbial intervention, are making strides towards clinical application. We highlight existing limitations and future directions for enhancing the quality of preclinical and clinical research. Efforts to decipher the complexities of pain pathology will be instrumental in translating scientific discoveries into clinical practice, thereby improving pain management from bench to bedside. 

Pathology of pain and its implications for therapeutic interventions

Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with gigaohm seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPR. Additionally, we tested the effect of 36 arthropod venoms on NaV 1.9 using a single 384-well plate on the SyncroPatch 384. As an example for mutant screening, a range of acid-sensing ion channel variants were tested and the success rate increased through fluorescence-activated cell sorting (FACS) prior to APC experiments. Gigaohm seal data quality makes the 384-format accessible to recording of primary and stem cell-derived cells on the SyncroPatch 384. We show recordings in voltage and current clamp modes of stem cell-derived cardiomyocytes. In addition, the option of intracellular solution exchange enabled investigations into the effects of intracellular Ca2+ and cAMP on TRPC5 and HCN2 currents, respectively. Together, these data highlight the broad applicability and versatility of APC platforms and also outlines some limitations of the approach. KEY POINTS: High throughput automated patch clamp (APC) can be used for a variety of applications involving ion channels. Lower false positive rates were achieved using automated patch clamp versus a fluorometric imaging plate reader (FLIPR) in a high throughput compound screen against NMDA receptors.  Genetic variants and mutations can be screened on a single 384-well plate to reduce variability of experimental parameters. Intracellular solution can be perfused to investigate effects of ions and second messenger systems without the need for excised patches. Primary cells and stem cell-derived cells can be used on high throughput APC with reasonable success rates for cell capture, voltage clamp measurements and action potential recordings in current clamp mode.

The suitability of high throughput automated patch clamp for physiological applications

Endogenous ion channels expressed in human embryonic kidney (HEK-293) cells

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics. Significance Statement Neuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way towards future analgesics.

https://www.biorxiv.org/content/biorxiv/early/2019/10/29/816264.full.pdf

Mechanism and site of action of big dynorphin on ASIC1a

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Mechanism and site of action of big dynorphin on ASIC1a.

Mechanism and site of action of big dynorphin on ASIC1a