Figure 4. H_v1 homologues in coccolithophores function as H⁺ channels.

(A) Whole cell currents in HEK293 cells transfected with EhHVCN1, CpHVCN1, and GFP only. The pipette solution contained (in mM) NMDG 65, MgCl₂ 3, EGTA 1, and HEPES 150 glucose 70 at pH 7.0 (P4, Table S2), and the bath solution contained (in mM) N-methyl D-glucamine (NMDG) 75, MgCl₂ 3, CaCl₂ 1, glucose 160, and HEPES 100 at pH 7.8 (E4, Table S2). Cells were depolarised in 10 mV increments from −60 to +100 mV. For clarity, every other trace is shown. (B) Average current-voltage curves (± SE, n = 6) of HEK293 cells transfected with EhHVCN1 (red circles), CpHVCN1 (blue squares), and GFP only (black triangles), respectively, using the protocol described in (A). (C) Tail current analysis of HEK293 cells expressing EhHVCN1. Currents were activated by a 500 ms depolarisation to +80 mV followed by repolarisation to between −50 and +70 mV. Dotted line represents zero current level. The solutions used for patch and bath solutions were the same as described in (A) (E4, P4). (D) pH dependence of EhH_v1 E_rev. (E) Outward currents of EhH_v1-expressing HEK 293 cells activated by depolarising pulses from −60 mV to +100 mV before (upper panel) and after (lower panel) perfusion with pH_o 6.5. The pipette solution was as in (A) and the external solutions were as described above except that 100 mM MES replaced 100 mM HEPES at pH 6.5 (E5, Table S2). (F) Average current-voltage curves (± SE, n = 5) for pH_o 7.8 or 6.5. Activation voltage shifts with E_H ⁺ (arrows). (G) Inhibition of EhH_v1 currents by 500 µM Zn²⁺. The recording pipette contained in mM: NaCl 30, KCL 100, MgCl₂ 3, EGTA 1, and HEPES 100 at pH 7.0 (P3, Table S2). Cells were bathed with (in mM) NaCl 160, KCL 2.0, MgCl₂ 1, CaCl₂ 1, Glucose 20, and HEPES 100 at pH 7.8 (E3, Table S2). (H) Average current voltage curves (± SE, n = 5) for control and in the presence of 500 µM Zn²⁺.