Principle obstacles for the transplantation of the K+ sensor from Kout to Kin channels

PAWEL GAJDANOWICZ; CARLOS GARCIA-MATA; BERND MUELLER-ROEBER; MICHAEL R. BLATT; INGO DREYER

Valencia, España

Workshop; XIV International Workshop on Plant Membrane Biology; 2007

In Arabidopsis, the nine Kv (Shaker-like voltage-gated) K+ channel proteins comprise a common structural framework of six transmembrane domains. However, the same channels show a remarkable functional diversity, segregating between at least three distinct subfamilies that include both inward-rectifying (Kin) and outward rectifying (Kout) channels. These plant Kin and Kout channels also differ in their sensitivity towards the extracellular K+ concentration. Whereas Kin channels are largely inert to K+ and react only to the transmembrane voltage, Kout channels sense the prevailing extracellular K+ concentration and open significantly only at membrane voltages when the driving force for net K+ flux is directed outward. Key structural determinants important for the K+ sensitivity are associated with the pore and the S6 helix. The functional difference between Kin and Kout channels is indeed intriguing. Although the gating characteristics of both channel subfamilies are clearly encoded within the channel protein structures, what features are critical to inward- and outward-rectification, and why gating is K+ sensitive in one case but not in the other is not obvious. In this study we tested whether the K+ sensor from Kout channels can be transplanted to Kin channels. A sequence alignment of the S6 _helices of Kin Kout rectifying K+ channel subfamilies highlights their close similarities, notably within the central and C-terminal regions of the helix. Over the stretch of 18 amino acid residues KAT1 and SKOR share 66% identity and retain virtually complete identity with other members of their respective subfamilies. We identified two discrete regions of the S6 helices that differ between the representatives KAT1 and SKOR. We show that these regions are important for gating in both channels, but that the two regions influence gating differently, demonstrating a lack of functional complementarity between the two K+ channels. We report (i) that mutations of the central and C-terminal domains influence the gating of both K+ channels, (ii) that in general exchange of the corresponding residues between KAT1 and SKOR does not yield complementary effects on gating, and (iii) that mutations of the central residues in SKOR appear dominant over those at the C-terminus, whereas the reverse is true for KAT1. We find that voltage- and K+-dependent gating of the outward-rectifier SKOR is most sensitive to mutations in a cluster of residues defining the S6 “gating domain”- dominating even lethal amino acid substitutions near the C-terminus of the S6 helix. The results allowed us to conclude that different gating mechanisms prevail in the two Kv channels, despite their close structural similarities and that this difference is a barrier for the transplantation of the K+-sensor from Kout to Kin channels.