Abstract
Acid-sensing ion channel 1a (ASIC1a) is a primary acid sensor in the peripheral and central nervous system. It has been implicated as a novel therapeutic target for a broad range of pathophysiological conditions including pain, ischemic stroke, depression, and autoimmune diseases such as multiple sclerosis. The only known selective blocker of ASIC1a is π-TRTX-Pc1a (PcTx1), a disulfide-rich 40-residue peptide isolated from spider venom. π-TRTX-Pc1a is an effective analgesic in rodent models of acute pain and it provides neuroprotection in a mouse model of ischemic stroke. Thus, understanding the molecular basis of the π-TRTX-Pc1a–ASIC1a interaction should facilitate development of therapeutically useful ASIC1a blockers. We therefore developed an efficient bacterial expression system to produce a panel of π-TRTX-Pc1a mutants for probing structure-activity relationships as well as isotopically labeled toxin for determination of its solution structure and dynamics. We demonstrate that the toxin pharmacophore resides in a β-hairpin loop that was revealed to be mobile over a wide range of time scales using molecular dynamics simulations in combination with NMR spin relaxation and relaxation dispersion measurements. The toxin-receptor interaction was modeled by in silico docking of the toxin structure onto a homology model of rat ASIC1a in a restraints-driven approach that was designed to take account of the dynamics of the toxin pharmacophore and the consequent remodeling of side-chain conformations upon receptor binding. The resulting model reveals new insights into the mechanism of action of π-TRTX-Pc1a and provides an experimentally validated template for the rational design of therapeutically useful π-TRTX-Pc1a mimetics.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Health and Medical Research Council of Australia [Project Grant 511067]; and the Australian Research Council [Discovery Grants DP0878608, DP0987043, DP0879065]. N.J.S. was supported by an Australian Postgraduate Award from the Australian Research Council. M.B. was supported by postdoctoral fellowships from the Swiss National Science Foundation [Fellowships PBBEP3-125613, PA00P3-134167].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.072207.
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ABBREVIATIONS:
- ASIC
- acid-sensing ion channel
- PcTx1
- Psalmopoeus cambridgei toxin-1 (psalmotoxin-1)
- π-TRTX-Pc1a
- π-theraphotoxin-Pc1a
- MBP
- maltose binding protein
- TEV
- tobacco etch virus
- IPTG
- isopropyl β-d-1-thiogalactopyranoside
- Ni-NTA
- nickel-nitrilotriacetic acid
- HPLC
- high-performance liquid chromatography
- rpHPLC
- reversed-phase high-performance liquid chromatography
- MALDI-TOF
- matrix-assisted laser desorption ionization/time of flight
- MS
- mass spectroscopy
- NUS
- nonuniform sampling
- HSQC
- heteronuclear single quantum correlation
- NOESY
- nuclear Overhauser effect (or enhancement) spectroscopy
- HNHB
- (amide proton)-(nitrogen)-(β-proton) correlation
- NOE
- nuclear Overhauser effect
- 3D
- three dimensional
- CPMG
- Carr-Purcell-Meiboom-Gill
- MD
- molecular dynamics
- ECD
- extracellular domain
- TM
- transmembrane
- PDB
- Protein Data Bank
- AIR
- ambiguous interaction restraint
- R1
- longitudinal relaxation rate
- R2
- transverse relaxation rate
- Rex
- chemical/conformational exchange rate constant.
- Received March 12, 2011.
- Accepted August 8, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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