Identification of Thr29 as a critical phosphorylation site that activates the human proton channel Hvcn1 in leukocytes.
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 pH(i) regulation. The "enhanced gating mode" appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-delta phosphorylation sites in the human voltage-gated proton channel, H(V)1 (Hvcn1), Thr(29) and Ser(97), 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-delta. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H(V)1 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 H(V)1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr(29) 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.
Musset
Boris
B
Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois 60612, USA.
Capasso
Melania
M
Cherny
Vladimir V
VV
Morgan
Deri
D
Bhamrah
Mandeep
M
Dyer
Martin J S
MJ
DeCoursey
Thomas E
TE
D019943:Amino Acid Substitution; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D020125:Mutation, Missense; D019255:NADPH Oxidases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000378:metabolism; D016897:Respiratory Burst / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D002273:Carcinogens; D004791:Enzyme Inhibitors; C510235:HVCN1 protein, human; D007211:Indoles; D007473:Ion Channels; D008301:Maleimides; D013912:Threonine; D019255:NADPH Oxidases; D051745:Protein Kinase C-delta; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate