We have recently cloned the eighth member of the ether-Æ`a-go-go potassium channel family in human, heag2 (Ju & Wray, 2002). The activation kinetics of this channel are different from the closely related, homologous heag1 channel, which shows 73 % identity at the amino acid level. In this study, we have investigated the molecular regions that are involved in determining the differences in activation kinetics between heag1 and heag2 channels.

For this, chimaeras between heag1 and heag2 were constructed by restriction digest and subsequent ligation. Chimaeras were made by swapping amino acids 1-137 in the intracellular N terminal region, which includes the PAS domain, or by swapping amino acids 138-549, which includes the central transmembrane regions S1-S6. Constructs were expressed in Xenopus oocytes, and two-electrode voltage-clamp recordings were made 2 days later at room temperature. Test potentials were then applied to 0 mV (500 ms duration) from a holding potential of -80 mV, and activation times were measured as the time from 20 % to 80 % of maximal current. For each chimaera, wild-type recordings were carried out in the same batch to allow for variations between batches of oocytes.

Chimaera I with amino acids 1-137 of heag2 replaced by those of heag1 showed an activation time (19.0 ± 2.1 ms, n = 10, mean ± S.E.M.) that was not significantly different from that for heag1 wild-type (20.1 ± 6.0 ms, n = 5) but significantly faster than for heag2 wild-type (59.7 ± 12.7 ms, n = 6, P < 0.05, Student’s unpaired t test). This indicates that amino acids contained within the region 1-137 are involved in determining differences in activation times between the two channels.

Chimaera II with amino acids 138-549 of heag2 replaced by those of heag1 displayed an activation time (21.1 ± 2.7 ms, n = 8) that was also not significantly different from that for heag1 wild-type (14.0 ± 3.1 ms, n = 7) but significantly faster than for heag2 wild-type (86.2 ± 9.5 ms, n = 4, P < 0.05). This suggests that amino acids within the region 138-549 are also involved in determining differences in activation times between heag1 and heag2.

Taken together, these results suggest that some amino acids within the intracellular N-terminal region (which includes the PAS domain), as well as some residues within the central region (which includes the membrane-spanning domains S1-S6), are involved in determining differences in activation kinetics between heag1 and heag2 channels. It seems likely that these effects on channel activation in heag occur because of an interaction between the N-terminal region with the membrane-spanning region.