Efficient and Flexible Synthesis of New Photoactivatable Propofol Analogs

Receptor Potential ◽

General Anesthetic ◽

Bioorthogonal Chemistry ◽

Functional Assays ◽

Pain Pathway ◽

Synthetic Routes ◽

Transient Receptor ◽

Proof Of Principle

Propofol is a widely used general anesthetic, which acts by binding to and modulating several neuronal ion channels. We describe the synthesis of photoactivatable propofol analogs functionalized with an alkyne handle for bioorthogonal chemistry. Such tools are useful for detecting and isolating photolabeled proteins. We designed expedient and flexible synthetic routes to three new diazirine-based crosslinkable propofol derivatives, two of which have alkyne handles. As a proof of principle, we show that these compounds activate heterologously expressed Transient Receptor Potential Ankyrin 1 (TRPA1), a key ion channel of the pain pathway, with a similar potency as propofol in fluorescence-based functional assays. This work demonstrates that installation of the crosslinkable and clickable group on a short nonpolar spacer at the para position of propofol does not affect TRPA1 activation, supporting the utility of these chemical tools in identifying and characterizing potentially druggable binding sites in propofolinteracting proteins.

TRPV1: Structure, Endogenous Agonists, and Mechanisms

International Journal of Molecular Sciences ◽

10.3390/ijms21103421 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3421 ◽

Cited By ~ 2

Author(s):

Miguel Benítez-Angeles ◽

Sara Luz Morales-Lázaro ◽

Emmanuel Juárez-González ◽

Tamara Rosenbaum

Keyword(s):

Ion Channel ◽

Binding Sites ◽

Receptor Potential ◽

Transient Receptor Potential Vanilloid ◽

Trpv1 Channel ◽

Field Of Study ◽

Transient Receptor ◽

Shed Light ◽

Made In

The Transient Receptor Potential Vanilloid 1 (TRPV1) channel is a polymodal protein with functions widely linked to the generation of pain. Several agonists of exogenous and endogenous nature have been described for this ion channel. Nonetheless, detailed mechanisms and description of binding sites have been resolved only for a few endogenous agonists. This review focuses on summarizing discoveries made in this particular field of study and highlighting the fact that studying the molecular details of activation of the channel by different agonists can shed light on biophysical traits that had not been previously demonstrated.

The Human Transient Receptor Potential Vanilloid Type 6 Distal Promoter Contains Multiple Vitamin D Receptor Binding Sites that Mediate Activation by 1,25-Dihydroxyvitamin D3 in Intestinal Cells

Molecular Endocrinology ◽

10.1210/me.2006-0031 ◽

2006 ◽

Vol 20 (6) ◽

pp. 1447-1461 ◽

Cited By ~ 147

Author(s):

Mark B. Meyer ◽

Makoto Watanuki ◽

Sungtae Kim ◽

Nirupama K. Shevde ◽

J. Wesley Pike

Keyword(s):

Vitamin D ◽

Receptor Binding ◽

Binding Sites ◽

Receptor Potential ◽

Intestinal Cells ◽

Transient Receptor Potential Vanilloid ◽

Dihydroxyvitamin D3 ◽

Transient Receptor ◽

Distal Promoter

Identification and characterization of two distinct calmodulin-binding sites in the Trpl ion-channel protein of Drosophila melanogaster

Biochemical Journal ◽

10.1042/bj3140497 ◽

1996 ◽

Vol 314 (2) ◽

pp. 497-503 ◽

Cited By ~ 66

Author(s):

Coral G. WARR ◽

Leonard E. KELLY

Keyword(s):

Protein Kinase ◽

Ion Channel ◽

Binding Sites ◽

Receptor Potential ◽

Free Form ◽

Calmodulin Binding ◽

Dependent Protein ◽

Two putative light-sensitive ion channels have been isolated from Drosophila, encoded by the transient-receptor-potential (trp) and transient-receptor-potential-like (trpl) genes. The cDNA encoding the Trpl protein was initially isolated on the basis that the expressed protein binds calmodulin. Using both fusion proteins and a synthetic peptide, we now show that two calmodulin-binding sites are present in the C-terminal domain of the Trpl protein, CBS-1 and CBS-2. CBS-1 binds calmodulin in a Ca2+-dependent fashion, requiring Ca2+ concentrations above 0.3–0.5 μM for calmodulin binding. In contrast, CBS-2 binds the Ca2+-free form of calmodulin, with dissociation occurring at Ca2+ concentrations between 5 and 25 μM. Phosphorylation of a serine residue within a peptide encompassing CBS-1 by cyclic AMP-dependent protein kinase (PKA) abolishes calmodulin binding, and phosphorylation of the adjacent serine by protein kinase C appears to modulate this phosphorylation by PKA. Interpretation of these findings provides a novel model for ion-channel gating and modulation in response to changing levels of intracellular Ca2+.

Modulation of Transient receptor potential melastatin 3 by protons through its intracellular binding sites

10.1101/2020.10.08.331454 ◽

2020 ◽

Author(s):

Md Zubayer Hossain Saad ◽

Liuruimin Xiang ◽

Yan-Shin Liao ◽

Leah R. Reznikov ◽

Jianyang Du

Keyword(s):

Binding Sites ◽

Pancreatic Beta Cells ◽

Outward Current ◽

Receptor Potential ◽

Ph Sensitivity ◽

Single Mutation ◽

Transient Receptor Potential Melastatin ◽

Intracellular Binding ◽

AbstractTransient receptor potential melastatin 3 channel (TRPM3) is a calcium-permeable nonselective cation channel that plays an important role in modulating glucose homeostasis in the pancreatic beta cells. However, how TRPM3 is regulated under physiological and pathological conditions is poorly understood. In this study, we found that both intracellular and extracellular protons block TRPM3 through its intracellular binding sites. We demonstrated that external protons indirectly block TRPM3, whereas internal protons inhibit TRPM3 directly with an inhibitory pH50 of 6.9 ± 0.11. We identified three titratable residues, D1059, D1062, and D1073, at the inner vestibule of the channel pore that contribute to pH sensitivity. The mutation of D1073Q reduces TRPM3 current intensity and pH sensitivity; Replacement of Asp 1073 by Gln 1073 changes the reduction of TRPM3 outward current by low external pH 5.5, from 62 ± 3 % in WT to 25 ± 6.0 % in D1073Q. These results indicate that D1073 is not only essential for intracellular pH sensitivity, but it is also crucial for TRPM3 channel gating. In addition, a single mutation of D1059 or D1062 enhances pH sensitivity. In summary, our findings provide a novel molecular determinant for pH regulation of TRPM3. The inhibition of TRPM3 by protons may indicate an endogenous mechanism governing TRPM3 gating and its physiological/ pathological functions.

Redox and trace metal regulation of ion channels in the pain pathway

Biochemical Journal ◽

10.1042/bj20150522 ◽

2015 ◽

Vol 470 (3) ◽

pp. 275-280 ◽

Cited By ~ 5

Author(s):

J. Grayson Evans ◽

Slobodan M. Todorovic

Keyword(s):

Ion Channels ◽

Trace Metal ◽

Redox Regulation ◽

Receptor Potential ◽

Transient Receptor Potential Channels ◽

Pain Pathway ◽

Alternative Means ◽

Potential Channels ◽

Given the clinical significance of pain disorders and the relative ineffectiveness of current therapeutics, it is important to identify alternative means of modulating nociception. The most obvious pharmacological targets are the ion channels that facilitate nervous transmission from pain sensors in the periphery to the processing regions within the brain and spinal cord. In order to design effective pharmacological tools for this purpose, however, it is first necessary to understand how these channels are regulated. A growing area of research involves the investigation of the role that trace metals and endogenous redox agents play in modulating the activity of a diverse group of ion channels within the pain pathway. In the present review, the most recent literature concerning trace metal and redox regulation of T-type calcium channels, NMDA (N-methyl-D-aspartate) receptors, GABAA (γ-aminobutyric acid A) receptors and TRP (transient receptor potential) channels are described to gain a comprehensive understanding of the current state of the field as well as to provide a basis for future thought and experimentation.

The capsaicin receptor

10.1093/med/9780198834359.003.0027 ◽

2018 ◽

Author(s):

Istvan Nagy

Keyword(s):

Ion Channel ◽

Receptor Potential ◽

Chilli Pepper ◽

Capsaicin Receptor ◽

Major Cation ◽

Starting Point ◽

Pain Pathway ◽

Transient Receptor ◽

Trp Ion Channel

The landmark paper discussed in this chapter is ‘The capsaicin receptor: A heat activated ion channel in the pain pathway’, published by Caterina et al. in 1997. The identification of the molecular basis for the sensitivity of a major proportion of nociceptive primary sensory neurons for capsaicin, the pungent agent in chilli pepper, was undoubtedly one of the most significant pain-related discoveries in the twentieth century, for at least three reasons. First, the mechanism for capsaicin-induced responses could unequivocally be explained. Second, the discovery heralded the starting point for the development of a highly promising, mechanism-based means of analgesia. Third, the discovery also sparked studies which resulted in the discovery of the major cation channel family, the transient receptor potential (TRP) ion channel family, several members of which have also become putative targets for the development of analgesics.

Phosphoinositides regulate the TRPV1 channel via two functionally distinct binding sites

10.1101/558874 ◽

2019 ◽

Author(s):

Aysenur Torun Yazici ◽

Eleonora Gianti ◽

Marina A. Kasimova ◽

Vincenzo Carnevale ◽

Tibor Rohacs

Keyword(s):

Binding Site ◽

Binding Sites ◽

Receptor Potential ◽

Transient Receptor Potential Vanilloid ◽

Trpv1 Channel ◽

Distinct Site ◽

Excised Patches ◽

Transient Receptor ◽

Dynamics Simulations

AbstractRegulation of the heat- and capsaicin-activated Transient Receptor Potential Vanilloid 1 (TRPV1) channel by phosphoinositides is controversial. In a recent cryoEM structure, an endogenous phosphoinositide was detected in the vanilloid binding site, and phosphoinositides were proposed to act as competitive vanilloid antagonists. This model is difficult to reconcile with phosphatidylinositol 4,5- bisphosphate [PtdIns(4,5)P2] being a well established positive regulator of TRPV1. To resolve this controversy, we propose that phosphoinositides regulate TRPV1 via two functionally distinct binding sites. Our molecular dynamics simulations show that phosphatidylinositol (PtdIns) is more stable in the vanilloid binding site, whereas a distinct site responsible for activation is preferentially occupied by PtdIns(4,5)P2. Consistently, we show that in the presence of PtdIns(4,5)P2 in excised patches PtdIns partially inhibited TRPV1 activity induced by low, but not high capsaicin concentrations. In the absence of PtdIns(4,5)P2 on the other hand, PtdIns partially stimulated TRPV1 activity presumably by binding to the activating site. Overall, our data resolve a major controversy in the regulation of TRPV1.

Druggable Lipid Binding Sites in Pentameric Ligand-Gated Ion Channels and Transient Receptor Potential Channels

Frontiers in Physiology ◽

10.3389/fphys.2021.798102 ◽

2022 ◽

Vol 12 ◽

Author(s):

Wayland W. L. Cheng ◽

Mark J. Arcario ◽

John T. Petroff

Keyword(s):

Ion Channels ◽

Ion Channel ◽

Binding Sites ◽

Receptor Potential ◽

Lipid Binding ◽

Transient Receptor Potential Channels ◽

Allosteric Modulators ◽

Potential Channels ◽

Lipids modulate the function of many ion channels, possibly through direct lipid-protein interactions. The recent outpouring of ion channel structures by cryo-EM has revealed many lipid binding sites. Whether these sites mediate lipid modulation of ion channel function is not firmly established in most cases. However, it is intriguing that many of these lipid binding sites are also known sites for other allosteric modulators or drugs, supporting the notion that lipids act as endogenous allosteric modulators through these sites. Here, we review such lipid-drug binding sites, focusing on pentameric ligand-gated ion channels and transient receptor potential channels. Notable examples include sites for phospholipids and sterols that are shared by anesthetics and vanilloids. We discuss some implications of lipid binding at these sites including the possibility that lipids can alter drug potency or that understanding protein-lipid interactions can guide drug design. Structures are only the first step toward understanding the mechanism of lipid modulation at these sites. Looking forward, we identify knowledge gaps in the field and approaches to address them. These include defining the effects of lipids on channel function in reconstituted systems using asymmetric membranes and measuring lipid binding affinities at specific sites using native mass spectrometry, fluorescence binding assays, and computational approaches.

Inositol lipids and TRPC channel activation

Biochemical Society Symposium ◽

10.1042/bss2007c04 ◽

2007 ◽

Vol 74 ◽

pp. 37-45 ◽

Cited By ~ 12

Author(s):

James W. Putney

Keyword(s):

Plasma Membrane ◽

Phospholipase C ◽

Calcium Signalling ◽

Receptor Potential ◽

Breakdown Product ◽

Channel Activation ◽

Inositol Lipids ◽

Transient Receptor ◽

Secondary Mechanism

The original hypothesis put forth by Bob Michell in his seminal 1975 review held that inositol lipid breakdown was involved in the activation of plasma membrane calcium channels or ‘gates’. Subsequently, it was demonstrated that while the interposition of inositol lipid breakdown upstream of calcium signalling was correct, it was predominantly the release of Ca2+ that was activated, through the formation of Ins(1,4,5)P3. Ca2+ entry across the plasma membrane involved a secondary mechanism signalled in an unknown manner by depletion of intracellular Ca2+ stores. In recent years, however, additional non-store-operated mechanisms for Ca2+ entry have emerged. In many instances, these pathways involve homologues of the Drosophila trp (transient receptor potential) gene. In mammalian systems there are seven members of the TRP superfamily, designated TRPC1–TRPC7, which appear to be reasonably close structural and functional homologues of Drosophila TRP. Although these channels can sometimes function as store-operated channels, in the majority of instances they function as channels more directly linked to phospholipase C activity. Three members of this family, TRPC3, 6 and 7, are activated by the phosphoinositide breakdown product, diacylglycerol. Two others, TRPC4 and 5, are also activated as a consequence of phospholipase C activity, although the precise substrate or product molecules involved are still unclear. Thus the TRPCs represent a family of ion channels that are directly activated by inositol lipid breakdown, confirming Bob Michell's original prediction 30 years ago.