Protein interactions central to stabilizing the K ⁺ channel selectivity filter in a four-sited configuration for selective K ⁺ permeation

Abstract

The structural and functional conversion of the nonselective NaK channel to a K + selective channel (NaK2K) allows us to identify two key residues, Tyr and Asp in the filter sequence of TVG Y G D , that participate in interactions central to stabilizing the K + channel selectivity filter. By using protein crystallography and channel electrophysiology, we demonstrate that the K + channel filter exists as an energetically strained structure and requires these key protein interactions working in concert to hold the filter in the precisely defined four-sited configuration that is essential for selective K + permeation. Disruption of either interaction, as tested on both the NaK2K and eukaryotic K v 1.6 channels, can reduce or completely abolish K + selectivity and in some cases may also lead to channel inactivation due to conformational changes at the filter. Additionally, on the scaffold of NaK we recapitulate the protein interactions found in the filter of the Kir channel family, which uses a distinct interaction network to achieve similar stabilization of the filter.

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

“…The structural stability of NaK has enabled engineering of the selectivity filter, tuning the number of ion binding sites and ion selectivity of the channel (21,23). Beautiful high-resolution crystal structures of these different mutants have contributed to our current understanding of the structure-function relationships underlying ion selectivity and support the model that four ion binding sites are necessary for potassium selectivity through a knock-on mechanism (21-23, 25, 31-33).…”

“…NaK constructs were missing the first 19 amino acids, corresponding to the NaKΔ19 construct used to determine most crystal structure of NaK and NaK mutants (21,23). NaKΔ19 is referred to as NaK throughout to emphasize the different mutations within this construct that are the focus of this work.…”

“…The NaK channel has the same basic pore architecture as K + channels ( Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence ( 63 TVGDGN 68 ) and structure (19)(20)(21)(22)(23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig.…”

“…However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence ( 63 TVGYGD 68 ; Fig. 1 A and B), leading to K + selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21,23). This K + -selective mutant of NaK is called NaK2K.…”

A structural, functional, and computational analysis suggests pore flexibility as the base for the poor selectivity of CNG channels

“…In K + channels, two key interactions involving the tyrosine residue in the selectivity filter and the charged aspartate immediately following the signature sequence play a major role in maintaining the conductive conformation of the filter and are necessary for K + selectivity (42,43). The ring of bulky tyrosine residues known as the "aromatic girdle" (4, 7) is conserved in most K + channels and is thought to provide the putative rigidity and the network of hydrogen bonds necessary to stabilize the structure of the selectivity filter (4,43,44). Moreover, the aspartate immediately following the filter has been shown to participate in a multipoint hydrogen bond network defining the energetic profile of C-type inactivation in KcsA channels (42,45,46).…”