Eag channels are members of the ether-a-go-go family of potassium channels, and the human eag2 (heag2) channel has been recently cloned and characterised by ourselves (Ju & Wray, 2002) and by Schonherr et al. (2002). We compared the steady-state activation properties of this channel with heag1, and found that, in normal potassium solutions, the conductance-voltage (G-V ) curve for heag2 is shifted to the left and shows less steep voltage dependence, compared with heag1. Molecular regions that might account for these functional differences may include the S4 transmembrane region or the nearby linkers. However, the S4 region and the S3-S4 linker are identical between the two channels, while there is only one amino acid that is different in the S4-S5 linker: isoleucine in heag1 at position 345 and leucine in heag2. Here we have investigated a possible role of this residue in steady-state activation.

For this, we made the mutation I345L in heag1, using PCR Quickchange mutagenesis. RNA for wild-type heag1 and mutant I345L was injected into Xenopus oocytes, and two-electrode voltage-clamp recordings were made at room temperature 2 days later. Cells were held at -80 mV, and depolarising pulses applied at 0.1 Hz (500 ms duration). Current-voltage recordings were made in normal potassium solutions, and Boltzmann curves were fitted to the corresponding G-V curves, parametrised in the usual way (slope parameter k, voltage for half-maximal activation V_0.5).

For the current-voltage curves, there were no significant differences (Student’s unpaired t test) between the currents for wild-type and I345L mutant heag1 channels for a range of test potentials from -30 to +50 mV. Consistent with this, the Boltzmann slope parameter, k, was not significantly different between the two channels (17.1 ± 1.4 mV, n = 6, for wild-type; 17.2 ± 1.5 mV for the mutant, n = 7, means ± S.E.M.); the V_0.5 parameter was also not significantly different (9.3 ± 2.1 mV for wild-type, 6.6 ± 1.5 mV for the mutant).

Therefore, the data show that I or L residues (present in heag1 and heag2, respectively) at position 345 in the S4-S5 linker are not involved in the molecular mechanism for the differences between steady-state activation for heag1 and heag2 channels. The result is perhaps not surprising because residues I and L are both amino acids with similar properties such as size, hydrophobicity and neutral charge.

However, residue 345 may be of importance in determining the kinetics of activation, which also differs between heag1 and heag2 channels. Further work is in progress therefore to investigate whether this residue in the S4-S5 linker is involved in activation kinetics.

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