Identification of Thr29 as a Critical Phosphorylation Site That Activates the Human Proton Channel Hvcn1 in Leukocytes

• Published in: The Journal of biological chemistry 2010-02, Vol.285 (8), p.5117-5121
• Main Authors: Musset, Boris, Capasso, Melania, Cherny, Vladimir V., Morgan, Deri, Bhamrah, Mandeep, Dyer, Martin J.S., DeCoursey, Thomas E.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Carcinogens - pharmacology; Cell Line; Cell/Phagocytosis; Channel/Other; Enzyme Inhibitors - pharmacology; Humans; Immunology/Innate Immunity; Indoles - pharmacology; Ion Channel Gating - drug effects; Ion Channel Gating - physiology; Ion Channels - genetics; Ion Channels - metabolism; Leukocytes - metabolism; Maleimides - pharmacology; Membrane/Channels; Mutation, Missense; NADPH Oxidases - metabolism; Phosphorylation; Phosphorylation - drug effects; Phosphorylation - physiology; Protein Kinase C-delta - antagonists & inhibitors; Protein Kinase C-delta - metabolism; Proton Channels; Proton Transport; Respiratory Burst - drug effects; Respiratory Burst - physiology; Signal Transduction; Tetradecanoylphorbol Acetate - pharmacology; Threonine - genetics; Threonine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.C109.082727

Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pHi regulation. The “enhanced gating mode” appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-δ phosphorylation sites in the human voltage-gated proton channel, HV1 (Hvcn1), Thr29 and Ser97, both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-δ. Enhanced gating was evaluated by expressing wild-type (WT) or mutant HV1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT HV1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr29 is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.