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Mutations in PKC Consensus Phosphorylation Sites Affect ASIC1 Channel Activity
Author(s) -
Bashari Edlira,
Anderson Susan J,
Fuller Catherine M,
Benos Dale J
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a535-d
Subject(s) - phosphorylation , protein kinase c , xenopus , patch clamp , amiloride , voltage clamp , chemistry , ion channel , electrophysiology , acid sensing ion channel , sodium channel , chelerythrine , microbiology and biotechnology , biophysics , biology , neuroscience , biochemistry , sodium , receptor , gene , organic chemistry
Acid‐sensing ion channel 1 (ASIC1) is a member of the Epithelial Na + Channel/Degenerin family. Both normal astrocytes and gliomas express ASIC1. However, only high‐grade gliomas exhibit an amiloride‐sensitive inward current. In patch‐clamp experiments, inclusion of PKCβ in the patch pipette inhibited this current, suggesting that PKC may phosphorylate ASIC1b, a potential component of the channel ( Berdiev et al. 2002 ). In this study, we mutated three consensus PKC phosphorylation sites (T26, S40, S499) in hASIC1b to A to prevent phosphorylation, and to E or D, to mimic phosphorylation. Using two‐electrode voltage clamp, we measured acid‐activated currents in Xenopus oocytes expressing ASIC1b. The T26A and T26E constructs did not exhibit acid‐activated currents, while S40A and S40E currents were indistinguishable from wt. In contrast, S499A and S499D exhibited currents that were 36.2 ± 22.4 % and 9.5 ± 9.9 % of wt. Application of the PKC activator PMA inhibited acid‐activated currents of wt hASIC1b to 69.3 ± 10% of the untreated control. PMA did not affect the currents associated with S40A, or S499A, and did not inhibit wt hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Our data suggest that S499 is a critical site mediating phosphorylation of hASIC1b. Further experiments will elucidate the impact of mutating this site in ASIC1 on glioma cell biology. This study was supported by NIH Grant CA101952.