Identification of Phosphorylation Sites that Activate Voltage Gated Proton Channels in Leukocytes

Abstract

One of the best established functions of proton channels is facilitating NADPH oxidase activity in phagocytes. NADPH oxidase moves electrons across the membrane, leaving protons in the cytoplasm. Proton channels extrude most of this acid, simultaneously compensating for the electrogenic action of the oxidase and preventing cytoplasmic acidification. Agonists that activate NADPH oxidase also produce an “enhanced gating mode” in proton channels, characterized by faster activation, increased conductance, and a negative shift of the gH-V relationship, all of which increase the likelihood of proton channel opening. The enhanced gating mode is the result of PKC activity (Morgan et al, 2007, J. Physiol. 579:327-344). We assessed proton channel responses in a murine B cell line, LK35.2. Nontransfected LK35.2 cells have no detectable proton current; cells transfected with the human proton channel gene, HVCN1, have proton currents that respond to PMA and the PKC inhibitor, GF109203X (GFX), when studied in perforated-patch configuration. The response of proton currents in these cells to PMA or GFX was qualitatively like that of native leukocytes, but smaller in amplitude. In contrast, there was no detectable PMA response of human or murine proton channels expressed in HEK-293 or COS-7 cells (Musset et al, 2008, J. Physiol. 586:2477-2486). Here we mutated two putative PKC phosphorylation sites on the human proton channel. When the mutant channels were expressed in LK35.2 cells, the response to PMA or GFX was ablated. These studies indicate that PKC phosphorylates the proton channel directly.Supported by the NIH-HLBI (HL61437), Philip Morris, and the Schmidtmann Foundation.