Double-Gap Omega-Currents in Shaker K-Channels used to Clarify the Activation Path of S4

Abstract

We have demonstrated previously for Shaker K-channels that the residues on the voltage sensor S4, namely A0(359) and the gating charges R1(362), R2(365) and R3(368) slide sequentially through the gating-pore (Gamal El-Din et al., 2010). Further, this gating pore was shown to encompass two of these residues since leak currents, so called omega-currents, appeared only when the two residues populating the pore were short, e.g. serine or alanine. We have studied the following mutants and found omega-currents for asRRR, RssRR and aRssR, while aRRss did not express so far (small letters denote short residues to visualize the double-gap position). A further sliding of S4 including R4(371) and K5(374) to the activated open state remained speculative.Here, we report further experiments on these double-gap mutants now combined with 434W instead of 434F to allow for K-current through the alpha-pore when S4 reaches the activated state. All three double-gap mutants show K-current which can be blocked by 4-AP confirming that S4 reaches the activated state. Interestingly, the mutant aRssR shows a voltage range of about 20 mV where neither omega- nor alpha-current is detected. This indicates that S4 steps from the leaking position with s2, s3 in the omega-pore through a next state with s3, R4 in the pore before reaching the activated state with R4, K5 in the pore. This interpretation goes well with the recent finding by Tao et al. (Science 2010) that an inner occluding binding site (for Shaker E293, F290, D316) is opposite to either residue K5 in the activated state or R1 in the closed state. In extension to this, our results demand an outer occluding site (most likely E283) opposed by R4 (activated) or a0 (closed), respectively.