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 domains (Frech et al. 1989; Albrecht et al. 1993). Despite this high degree of similarity, the activation rate of rat Kv2.1 is much faster than for the human channel. We have already demonstrated that N terminal residues play a role in activation kinetics (Ormond et al. 2001). In the C-terminal region, there are 49 amino acids that are different between the two channels, and here we have investigated a possible role of C-terminal residues in determining activation kinetics.

For this, cDNA chimaeras were made between rat and human forms of the Kv2.1 channel. These were constructed either by cutting with restriction enzymes and subsequent ligation of fragments, or by using the overlap extension PCR method. cRNAs for these chimaeras and for the wild-type channels were injected into Xenopus oocytes, and voltage-clamp recordings were made 1-2 days later at room temperature. Depolarizing pulses were applied at 0.1 Hz from a holding potential of -80 mV, and activation times were measured from 10 to 90 % maximal current.

The first chimaera with amino acids 108-740 of the rat channel replaced by the corresponding human channel showed an activation time (46 ± 4 ms at 0 mV, n = 4, mean ± S.E.M.) that was not significantly different (Student’s unpaired t test) from that for wild-type rat channel (48 ± 6 ms, n = 10). This indicates that amino acids 108-740 are not likely to play a part in activation kinetics. On the other hand, a second chimaera with amino acids 596-853 of the rat channel replaced by human sequence displayed a significantly (P < 0.05) longer activation time (92 ± 20 ms, n = 9) than that for the rat channel (42 ± 15 ms, n = 5), and in fact was similar to the activation time for human wild-type channel. The results for these two chimaeras therefore suggest that amino acids 741-853 probably contain a domain that plays a role in activation kinetics. A third chimaera was made to further define this domain, with amino acids 741-796 of the rat channel replaced by the corresponding human sequence. For this chimaera, the activation time (62 ± 8 ms, n = 6) was not significantly different from that for rat wild-type (54 ± 15 ms, n = 5). Taken together, these results indicate that a region spanning amino acids 797-853 of the C-terminal region contains a domain that plays a role in determining the activation kinetics of the Kv2.1 channel.

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