Role of the S2 and S3 segment in determining the activation kinetics in Kv2.1 channels

We constructed chimeras between the rapidly activating Kv1.2 channel and the slowly activating Kv2.1 channel in order to study to what extent sequence differences within the S1-S4 region contribute to the difference in activation kinetics. The channels were expressed in Xenopus oocytes and the currents were measured with a two-microelectrode voltage-clamp technique. Substitution of the S1-S4 region of Kv2.1 subunits by the ones of Kv1.2 resulted in chimeric channels which activated more rapidly than Kv2.1. Furthermore, activation kinetics were nearly voltage-independent in contrast to the pronounced voltage-dependent activation kinetics of both parent channels. Systematic screening of the 51-54 region by the replacement of smaller protein parts resolved that the main functional changes generated by the 51-54 substitution were generated by the 52 and the 53 segment. However, the effects of these segments were different: The 53 substitution reduced the effective gating charge and accelerated both a voltage-dependent and a voltage-independent component of the activation time course. In contrast. the 52 substitution accelerated predominantly the voltage-dependent component of the activation time course thereby leaving the effective gating charge unchanged. It is concluded that the 52 and the 53 segment determine the activation kinetics in a specific manner.