Equilibria between the K^+ binding and cation vacancy conformations of potassium channels

Dear Editor, Potassium channels are integral membrane proteins that selectively con duct K^+ ions across cell membra ne (Hille, 2001). They play essential roles in maintaining cellular ionic homoeostasis and generating action membrane potentials in excitable cells. The mechanism of K^+ selectivity has been evaluated in many biophysical and physiological studies (Zhou et al., 2001;Liu et al., 2015;Schewe et al., 2016). A highly con served signature seque nee, TVGYG, in the selectivity filter of potassium channels (e.g., KcsA) is known to coordinate K^+ ions (Zhou et al., 2001). Carbonyls of these residues point toward the pore, forming four continuous ion binding sites (S1-S4) and resulting in higher selectivity for K^+ over Na^+(Zhou et al., 2001). The NaK channel from Bacillus cereus is a norvselective cation channel that shares high structural homology with KcsA (Shi et al., 2006). Owing to a distinctive primary sequenee of 63TVGDG67, the selectivity filter of NaK preserves only two ion binding sites, allocated similarly as S3 and S4 in KcsA (Alam and Jiang, 2009a, b). Remarkably, the D66Y and N68D double mutations of NaK channel (Fig. S1) transform it into a K^+ selective channel (termed as NaK2K)(Sauer et al., 2013). Crystal structure of NaK and NaK2K have revealed distinet binding coordination of Na^+ and K^+ ions in their selectivity filter (Figs. S2 and S3)(Alam and Jiang, 2009a, b;Sauer et al., 2013). However, dynamics of the NaK and NaK2K selectivity filter with the bind泊g of Na+ or K^+ are still elusive. Especially, it has been known that the membrane environment is highly diverse from deterge nt micelles, which was con sidered to in flue nee the structure and functi on of membrane protei ns dramatically (Cross et al., 2011). Thus, it is necessary to study the cation and binding properties of NaK NaK2K channels in lipid bilayers.