Abstract
Voltage-gated potassium channels (Kv) are targets for drugs of large chemical diversity. Although hydrophobic cations block Kv channels with Hill coefficients of 1, uncharged electron-rich (cationophilic) molecules often display Hill coefficients of 2. The mechanism of the latter block is unknown. Using a combination of computational and experimental approaches, we mapped the receptor for the immunosuppressant PAP-1 (5-(4-phenoxybutoxy)psoralen), a high-affinity blocker of Kv1.3 channels in lymphocytes. Ligand-docking using Monte Carlo minimizations suggested a model in which two cationophilic PAP-1 molecules coordinate a K+ ion in the pore with their coumarin moieties, whereas the hydrophobic phenoxyalkoxy side chains extend into the intrasubunit interfaces between helices S5 and S6. We tested the model by generating 58 point mutants involving residues in and around the predicted receptor and then determined their biophysical properties and sensitivity to PAP-1 by whole-cell patch-clamp. The model correctly predicted the key PAP-1-sensing residues in the outer helix, the P-loop, and the inner helix and explained the Hill coefficient of 2 by demonstrating that the Kv1.3 pore can accommodate two or even four PAP-1 molecules. The model further explained the voltage-dependence of block by PAP-1 and its thousand-fold selectivity for Kv1.3 over non-Kv1 channels. The 23- to 125-fold selectivity of PAP-1 for Kv1.3 over other Kv1 channels is probably due to its preferential affinity to the C-type inactivated state, in which cessation of K+ flux stabilizes the tripartite PAP-1:K+:PAP-1 complex in the pore. Our study provides a new concept for potassium channel block by cationophilic ligands.
Footnotes
B.S.Z. and H.W. are joint senior authors.
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the Natural Sciences and Engineering Research Council of Canada; Canadian Institutes of Health Research [Grant MOP-53229]; the National Institutes of Health National Institute of General Medical Sciences [Grant R01-GM076063]; and a predoctoral fellowship from the Howard Hughes Medical Institutes. The 800 MHz NMR experiments were made possible by the National Science Foundation [Grant DBI722538].
Computations were made possible by the facilities of the Shared Hierarchical Academic Research Computing Network (available at http://www.sharcnet.ca).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.064014.
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ABBREVIATIONS:
- Kv
- voltage-gated potassium channel
- MC
- Monte Carlo
- MCM
- Monte Carlo minimization
- TEA
- tetraethylammonium
- k
- linkers L45
- o
- outer helix
- p
- a P-loop
- i
- inner helix
- AS-85
- 5-[4-(4-phenoxyphenoxy)butoxy]psoralen
- SB4
- 1,2-dihydro-N-methyl-4-(4-phenoxybutoxy)quinolin-2-one
- SB9
- 5-(4-phenoxybutoxy)-2H-[1]benzopyran-2-one
- SB7
- 9-(phenoxybutoxy)-9H-dibenzo[b,e]pyran
- SB23
- 1-(4-phenoxybutoxy)anthraquinone
- SB24
- 9-(4-phenoxybutoxy)phenanthrene
- PAP-1
- 5-(4-phenoxybutoxy)psoralen
- PAPS
- 4-(4-phenoxybutoxy)-7H-furo[3,2-g]chromene-7-thione
- MK-499
- (+)-N-[1′ (6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2,4′-piperidin)-6-yl]methanesulfonamide] monohydrochloride
- TRAM-34
- 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole.
- Received February 8, 2010.
- Accepted June 30, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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