The human and rat forms of the Kv2.1 potassium channel have identical amino acids over the membrane-spanning regions, but differ only in the N- and C-terminal chains (Frech et al. 1989; Albrecht et al. 1993). However, the activation of rat Kv2.1 is much faster than for the human channel. In this study, we have investigated a possible molecular basis for this difference in activation rate. In the N-terminal region, there are only two amino acids that are different between the rat and human channels (or possibly six are different: ambiguity arises from nearby potential start methionines in the human). Here, we have investigated a possible role of N-terminal residue 67 in the rat channel where glutamine (Q) is found, whereas the corresponding residue in the human channel is glutamate (E).

For this, cDNA mutants were engineered corresponding to rat Kv2.1 Q67E and human Kv2.1 E67Q, using standard PCR-based techniques. cRNA for these mutants and for wild-type channel was injected into Xenopus oocytes, and two-electrode voltage-clamp recordings made 1-2 days later at room temperature.

Depolarising pulses were applied at 0.1 Hz from a holding potential of -80 mV. The current-voltage curves for wild-type and mutant channels were not significantly different from each other whether for rat or for human, as previously observed for the wild-type channels (Leadbitter & Wray, 2000). For the human Kv2.1 channel, the mutation at residue 67 did not significantly affect the 10-90 % activation time, at test potentials of 0 to 70 mV (e.g. at 0 mV, 164 ± 20 ms, n = 13 wild-type; 164 ± 15 ms, n = 20 mutant, means ± S.E.M.). On the other hand, for the rat Kv2.1 channel, the mutation caused a slowing of activation at 0 to 70 mV (e.g. at 0 mV, 101 ± 6 ms, n = 13 wild-type; 156 ± 15 ms, n = 6 mutant, P < 0.05, t test). Furthermore, the activation time was not significantly different between that for the rat mutant channel and that for both human wild-type and human mutant channels.

These results suggest that residue 67, located in the N-terminal region, is of key importance in determining the activation kinetics of the Kv2.1 channel. However, the fact that the rat channel is affected by mutation at this site, but not the human channel, suggests that another interacting site may also be involved, probably located at the C-terminal region. The replacement of the neutral glutamine residue by the negatively charged glutamate may interfere electrostatically with such an interaction.

We thank The Wellcome Trust for support.

Albrecht, B., Lorra, C., Stocker, M. & Pongs, O. (1993). Receptors and Channels 1, 99-110.

Frech, G.C., VanDongen M.J., Schuster, G., Brown, A.M. & Joho, R.H. (1989). Nature 340, 642-645.

Leadbitter, E. & Wray, D. (2000). J. Physiol. 527.P, 122P.