The human ether-a-go-go related gene (HERG) has unusual kinetics with slow activation but very rapid and voltage-dependent inactivation. HERG inactivation is affected by mutations in the outer mouth region of the channel. The outer pore region of HERG K⁺ channels has a much longer S5-P linker than other voltage-gated K⁺ channels and contains an amphipathic α-helix that modulates inactivation (Liu et al. 2002; Torres et al. 2003). The rat EAG-like (ELK) channel (RELK2) possesses a similarly long S5-P linker but has markedly reduced C-type like inactivation in comparison to HERG channels. To investigate the molecular basis of these differences we made chimaeras between HERG and RELK2 and studied their inactivation properties in Xenopus oocytes using standard inactivation protocols (Torres et al. 2003). All data are mean ± S.E.M. The V_0.5 of inactivation of RELK2 channels was 31.8 ± 3.4 mV (n=5) compared to V_0.5 of -70.3 ± 6.1 mV (n=6) for WT HERG. A chimaeric channel consisting of HERG but with the α-helix from the S5-P region of RELK2 (HERG/ELK S5P) had inactivation characteristics resembling WT RELK2 channels (V_0.5 of 27.3 ± 0.3 mV, n=4). This confirms the importance of this region in inactivation. Whilst the structure of the amphipathic helix in the S5-P linker is predicted to be the same in the HERG/RELK2 S5-P chimaera as WT HERG and the hydrophobic residues in this segment are highly conserved, there are significant variations in the hydrophilic residues. N588 in HERG is replaced with a glutamate in RELK2 and D591 and Q592 are replaced with arginines. Mutation of HERG N588E resulted in channels that rapidly inactivated with a V_0.5 of inactivation of -109 ± 5.2 mV (n=5). The HERG D591R/Q592R double mutant displayed current characteristics similar to both WT RELK2 and the S5-P chimaera with a V_0.5 of inactivation of 28.7 ± 3.3 mV (n=5). Mutation of both HERG N588E and D591R/Q592R together led to a channel with functional characteristics similar to D591R/Q592R alone (V_0.5 of inactivation of 26.9 ± 1.7 mV (n=5)), highlighting the severity of inactivation disruption with the presence of positive charges on the S5-P loop. A simple model for the role of the S5-P in inactivation that is consistent with our data is that the S5-P amphipathic helix interacts with a positively charged region elsewhere on the channel. Consequently, the addition of negative charge (e.g. N588E) enhances inactivation, whereas the addition of positive charge (e.g. D591R/Q592R) inhibits inactivation.