Evolving potassium channels by means of yeast selection reveals structural elements important for selectivity

Abstract

Potassium channels are widely distributed. To serve their physiological functions, such as neuronal signaling, control of insulin release, and regulation of heart rate and blood flow, it is essential that K ؉ channels allow K ؉ but not the smaller and more abundant Na ؉ ions to go through. The narrowest part of the channel pore, the selectivity filter formed by backbone carbonyls of the GYGcontaining K ؉ channel signature sequence, approximates the hydration shell of K ؉ ions. However, the K ؉ channel signature sequence is not sufficient for K ؉ selectivity. To identify structural elements important for K ؉ selectivity, we randomly mutagenized the G protein-coupled inwardly rectifying potassium channel 3.2 (GIRK2) bearing the S177W mutation on the second transmembrane segment. This mutation confers constitutive channel activity but abolishes K ؉ selectivity and hence the channel's ability to complement the K ؉ transport deficiency of ⌬trk1⌬trk2 mutant yeast. S177W-containing GIRK2 mutants that support yeast growth in low-K ؉ medium contain multiple suppressors, each partially restoring K ؉ selectivity to S177W-containing double mutants. These suppressors include mutations in the first transmembrane segment and the pore helix, likely exerting long-range actions to restore K ؉ selectivity, as well as a mutation of a second transmembrane segment residue facing the cytoplasmic half of the pore, below the selectivity filter. Some of these suppressors also affected channel gating (channel open time and opening frequency determined in single-channel analyses), revealing intriguing interplay between ion permeation and channel gating.

Electrostatic interactions in the channel cavity as an important determinant of potassium channel selectivity

“…Here, biology may echo engineering in instigating more than one mechanism to ensure the robustness of a desired outcome. Although discussions of evolution by nature are speculative, these considerations are in line with the results from our previous molecular evolution experiments (15) and the experimental verification of predictions presented in this study.…”

“…With this nonselective channel as a starting point, we sought to define determinants of K ϩ selectivity by using molecular evolution and searched for second-site suppressors that restore K ϩ selectivity to the S177W mutant channel. We identified several second-site suppressors, all outside the selectivity filter, revealing previously unidentified structural elements that are important for K ϩ selectivity and suggesting that selectivity may not be controlled solely by the selectivity filter (15). By following up on a particular second-site suppressor in the channel central cavity, we now demonstrate that K ϩ selectivity can be regulated through judicious engineering of cavity residues in Kir3.2 channels.…”

“…Mutations outside the selectivity filter, including positions in the transmembrane helix (14,15) and the cytoplasmic domain (16), have been shown to affect selectivity, although the underlying mechanism is unknown. In a previous study, we identified mutations in the pore-lining inner M2 segment of Kir3.2 [a G protein-activated inwardly rectifying potassium (Kir) channel], such as the S177W mutation, that greatly reduce the channel's ability to discriminate between K ϩ and Na ϩ (15).…”

“…In a previous study, we identified mutations in the pore-lining inner M2 segment of Kir3.2 [a G protein-activated inwardly rectifying potassium (Kir) channel], such as the S177W mutation, that greatly reduce the channel's ability to discriminate between K ϩ and Na ϩ (15). With this nonselective channel as a starting point, we sought to define determinants of K ϩ selectivity by using molecular evolution and searched for second-site suppressors that restore K ϩ selectivity to the S177W mutant channel.…”

“…1B), even though these channels still bear the intact K ϩ channel signature sequence. Starting from this nonselective channel, we used the molecular evolution approach to identify several mutations, all within the transmembrane domains, that restore selectivity to the double mutant channel bearing the S177W mutation (15). In particular, the N184D mutation of the M2 inner helix completely restores K ϩ selectivity (Fig.…”