Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K ⁺ channels

Abstract

K+ channels distinguish K + from Na + in the selectivity filter, which consists of four ion-binding sites (S1-S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of Ctype inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K + binding at the S2 site. We also show that an ester substitution at the S2 site in the K v AP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K + channels in K + compared with Rb + are due to different ion occupancies at the S2 site.

Individual Ion Binding Sites in the K+ Channel Play Distinct Roles in C-type Inactivation and in Recovery from Inactivation

“…(Dawson et al, 1994) Recently, we applied this backbone mutagenesis approach to alter ion occupancy at the S1 and S2 sites to investigate their roles in inactivation. (Matulef et al, 2013) We found that decreasing ion occupancy at the S1 site with the G79ester substitution did not affect the rate of inactivation, while decreasing occupancy at the S2 site with the Y78ester substitution nearly eliminated inactivation. We also found that the G77ester mutation nearly eliminated inactivation.…”

“…(Kent, 1988) We initially attempted semisynthesis of the V76ester channel using the previously reported two-part approach. (Matulef et al, 2013; Valiyaveetil et al, 2004) For introducing the ester substitution using semisynthesis, the ester linkage is initially incorporated into a synthetic peptide, which is then used for the assembly of the KcsA channel. The two part semisynthesis calls for the incorporation of the V76ester linkage into a 55 amino acid long peptide.…”

“…(Matulef et al, 2013; Valiyaveetil et al, 2006b) To improve the yields, we tested alternate coupling reagents and identified COMU (1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylaminomorpholinomethylene)]-methanaminium-hexafluorophosphate) as the optimal coupling reagent for introducing the ester linkage. (Twibanire and Grindley, 2011) This modification enabled synthesis of the V76ester pore peptide in good yields.…”

“…We have previously demonstrated that the 3′ amide-to-ester substitution (G77ester) impairs the ability of the channel to undergo C-type inactivation. (Matulef et al, 2013) The 3′ carbonyl oxygen contributes to both the S2 and S3 sites. To determine the effect of the 3′ ester substitution on the ion occupancies at these sites, we solved the structure of the KcsA G77ester channel.…”

“…5a). (Matulef et al, 2013) We determined the crystal structure of the KcsA G77ester and refined it to 2.3 Å resolution. The electron density corresponding to the selectivity filter is shown in Fig.…”