Identification of Thr29 as a critical phosphorylation site that activates the human proton channel Hvcn1 in leukocytes

Abstract

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.

FIGURE 1.

Identification of candidate phosphorylation sites in the human proton channel. A, location of the two putative PKC-δ phosphorylation sites in the sequence of H_V1. Both predicted PKC-δ sites are in the N terminus, with Ser⁹⁷ near the membrane boundary. Boxed residues indicate helical transmembrane regions (37). B, phosphorylation of H_V1 WT, T29A, S97A, and T29A/S97A mutants in the presence of recombinant PKC-δ and [γ-³²P]ATP in an in vitro kinase assay. PKC-δ was activated with 1 μm PMA. The myc immunoblot indicates loading. EV indicates empty vector control. The graph on the right-hand side represents densitometric analysis of the ³²P band versus loading control of 3–4 separate experiments (p < 0.05 for each mutant versus WT by Student's t test). Error bars indicate S.E.

FIGURE 2.

Enhanced proton channel gating elicited by PMA and reversed by GFX in an LK35.2 cell. A–C, families of proton currents during pulses in 10-mV increments up to +40 mV in an Hvcn1-transfected LK35.2 cell before (A) or after PMA stimulation (B) and after GFX (C). D, the steady-state g_H, calculated from the currents in A (■), B (○), and C (▵) after fitting to a rising exponential, using reversal potentials measured in each solution. E, τ_act values in the same cell are faster after PMA, but the effect is reversed by GFX. F, time course of PMA and GFX responses in the cell in A–E, with test pulses to +10 mV repeated every 38 s from a holding potential of −40 mV. During the break in the time course, after the addition of PMA, the family of currents in B was recorded. G, proton currents during pulses to +20 mV repeated every 20 s from a holding potential of −40 mV in an LK35.2 cell transfected with the T29A mutant channel did not respond to PMA or GFX.