Figure 6.
Figure 6. Role of the Elk1 N-terminal eag domain in mode shift. (A) Sequence alignment of the first 15 amino acids of the Cap domain for all human EAG family channels and fly (Dm) and Nematostella (Nv; sea anemone) Elk orthologues. Basic residues are shaded in blue, and other residues conserved in at least eight sequences are shaded in black. A universally conserved basic residue (K5 in Elk1) is highlighted with the red box. (B and C) On-cell current traces recorded in response to 1-s steps (−120 to 60 mV in 20-mV increments; −100-mV holding potential) for Elk1 Δ2–136 and K5Q. (D) Normalized on-cell tail currents recorded at −100 mV after 1-s prepulse to 60 mV for Δ2–136 and K5Q compared with WT (dotted line, zero current). (E and F) On-cell G-V curves recorded from a −100-mV hold (squares) or with a 1-s 60-mV prepulse (circles) for Δ2–136 and K5Q. (G) Normalized Δ2–136 and K5Q tail currents recorded at −100 mV after a 1-s prepulse to 60 mV from excised patches before and after the application of 10 µM PIP2. (H and I) G-V curves for Δ2–136 and K5Q determined from isochronal tail currents at −100 mV after 1-s steps to the indicated voltages for excised controls (red) and 10 µM PIP2 (blue) from a holding potential of −100 mV (squares) or with a 1-s prepulse to 60 mV (circles). (J) Mode shift values for Δ2–136 and K5Q compared with WT (*, P < 0.05; ***, P < 0.001; t test), on cell (black), excised (red), and plus 10 µM PIP2 (blue). (K) PIP2-dependent mode shift (ΔΔV50, 10 µM PIP2-exised control) for WT, K5Q, and Δ2–136 (***, P < 0.001 vs. WT; t test). (L) On-cell G-V curves for K5Q controls and +CiVSP recorded from a −100-mV hold (squares) or with a 1-s 60-mV prepulse (circles). (M) Mode shift measured on cell with or without CiVSP coexpression for K5Q compared with WT (***, P < 0.001; t test). All data are mean ± SEM; sample numbers are provided in Tables 1–3, and curves in G-V panels show single Boltzmann fits. GenBank accession numbers for the full sequences aligned in A are: Elk1, NM_144633; Elk2, NM_012284; Elk3, NM_012285; Eag1, NM_172362; Eag2, NM_139318; Erg1, NM_000238; Erg2, NM_030779; Erg3, NM_033272; DmElk, NM_057661; NvElk, KM_052387.

Role of the Elk1 N-terminal eag domain in mode shift. (A) Sequence alignment of the first 15 amino acids of the Cap domain for all human EAG family channels and fly (Dm) and Nematostella (Nv; sea anemone) Elk orthologues. Basic residues are shaded in blue, and other residues conserved in at least eight sequences are shaded in black. A universally conserved basic residue (K5 in Elk1) is highlighted with the red box. (B and C) On-cell current traces recorded in response to 1-s steps (−120 to 60 mV in 20-mV increments; −100-mV holding potential) for Elk1 Δ2–136 and K5Q. (D) Normalized on-cell tail currents recorded at −100 mV after 1-s prepulse to 60 mV for Δ2–136 and K5Q compared with WT (dotted line, zero current). (E and F) On-cell G-V curves recorded from a −100-mV hold (squares) or with a 1-s 60-mV prepulse (circles) for Δ2–136 and K5Q. (G) Normalized Δ2–136 and K5Q tail currents recorded at −100 mV after a 1-s prepulse to 60 mV from excised patches before and after the application of 10 µM PIP2. (H and I) G-V curves for Δ2–136 and K5Q determined from isochronal tail currents at −100 mV after 1-s steps to the indicated voltages for excised controls (red) and 10 µM PIP2 (blue) from a holding potential of −100 mV (squares) or with a 1-s prepulse to 60 mV (circles). (J) Mode shift values for Δ2–136 and K5Q compared with WT (*, P < 0.05; ***, P < 0.001; t test), on cell (black), excised (red), and plus 10 µM PIP2 (blue). (K) PIP2-dependent mode shift (ΔΔV50, 10 µM PIP2-exised control) for WT, K5Q, and Δ2–136 (***, P < 0.001 vs. WT; t test). (L) On-cell G-V curves for K5Q controls and +CiVSP recorded from a −100-mV hold (squares) or with a 1-s 60-mV prepulse (circles). (M) Mode shift measured on cell with or without CiVSP coexpression for K5Q compared with WT (***, P < 0.001; t test). All data are mean ± SEM; sample numbers are provided in Tables 1–3, and curves in G-V panels show single Boltzmann fits. GenBank accession numbers for the full sequences aligned in A are: Elk1, NM_144633; Elk2, NM_012284; Elk3, NM_012285; Eag1, NM_172362; Eag2, NM_139318; Erg1, NM_000238; Erg2, NM_030779; Erg3, NM_033272; DmElk, NM_057661; NvElk, KM_052387.

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