Engineering K+ Channels Using Semisynthesis

Potassium channels conduct K⁺ ions selectively and at very high rates. Central to the function of K⁺ channels is a structural unit called the selectivity filter. In the selectivity filter, a row of four K⁺ binding sites are created using mainly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, site-directed mutagenesis is of limited utility in investigating the selectivity filter. In order to overcome this limitation, we have developed a semisynthetic approach, which permits the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that we have developed for the semisynthesis of the K⁺ channel, KcsA. We anticipate that the protocols described in this chapter will also be applicable for the semisynthesis of other integral membrane proteins of interest.