Abstract
P2X ion channels have been functionally characterized from a range of eukaryotes. Although these receptors can be broadly classified into fast and slow desensitizing, the molecular mechanisms underlying current desensitization are not fully understood. Here, we describe the characterization of a P2X receptor from the cattle tick Boophilus microplus (BmP2X) displaying extremely slow current kinetics, little desensitization during ATP application, and marked rundown in current amplitude between sequential responses. ATP (EC50, 67.1 μM) evoked concentration-dependent currents at BmP2X that were antagonized by suramin (IC50, 4.8 μM) and potentiated by the antiparasitic drug amitraz. Ivermectin did not potentiate BmP2X currents, but the mutation M362L conferred ivermectin sensitivity. To investigate the mechanisms underlying slow desensitization we generated intracellular domain chimeras between BmP2X and the rapidly desensitizing P2X receptor from Hypsibius dujardini. Exchange of N or C termini between these fast- and slow-desensitizing receptors altered the rate of current desensitization toward that of the donor channel. Truncation of the BmP2X C terminus identified the penultimate residue (Arg413) as important for slow desensitization. Removal of positive charge at this position in the mutant R413A resulted in significantly faster desensitization, which was further accentuated by the negatively charged substitution R413D. R413A and R413D, however, still displayed current rundown to sequential ATP application. Mutation to a positive charge (R413K) reconstituted the wild-type phenotype. This study identifies a new determinant of P2X desensitization where positive charge at the end of the C terminal regulates current flow and further demonstrates that rundown and desensitization are governed by distinct mechanisms.
Footnotes
This work was supported by the Wellcome Trust [Grant WT081601MA] and a Biotechnology and Biological Sciences Research Council Studentship (to S.B.).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/mol.110.070037.
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ABBREVIATIONS:
- BmP2X
- P2X receptor from Boophilus microplus
- HdP2X
- P2X receptor from Hypsibius dujardini
- SmP2X
- P2X receptor from Schistosoma mansoni
- I20
- percentage of peak current amplitude after 20 s of ATP application
- T.10%
- time taken for peak current to decay by 10%
- PCR
- polymerase chain reaction
- TM
- transmembrane
- WT
- wild type
- EST
- expressed sequence tag.
- Received November 16, 2010.
- Accepted January 6, 2011.
- U.S. Government work not protected by U.S. copyright
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