Structure, function, and ion-binding properties of a K ⁺ channel stabilized in the 2,4-ion–bound configuration

Abstract

Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion–bound configuration (i.e., water, K+, water, K+-ion–bound configuration) of the K+ channel’s selectivity filter. A full functional and thermodynamic characterization of the G77A mutant revealed wild-type–like ion selectivity and apparent K+-binding affinity, in addition to showing a lack of C-type inactivation gating and a marked reduction in its single-channel conductance. These structures validate, from a structural point of view, the notion that 2 isoenergetic ion-bound configurations coexist within a K+ channel’s selectivity filter, which fully agrees with the water–K+-ion–coupled transport detected by streaming potential measurements.

TOK channels use the two gates in classical K + channels to achieve outward rectification

“…Two models for ion occupancy of these sites have been proposed to explain pore-filter control of permeation and inactivation. In the "canonical" model that we favor, ion occupancy in the pore alternates between two iso-energetic configurations where S1/S3 sites are occupied by K + ions (with S2/S4 sites occupied by water), or with K + ions in the S2/S4 sites 42,43 ; this model is supported by structural studies, [43][44][45][46][47] molecular dynamics simulations, 48 and streaming potential measurements, [49][50][51]…”

“…Arguing against this model, a recently published structure of KcsA with a T75A mutation that disrupts the S4 binding site, preventing all four sites from being occupied simultaneously, was demonstrated to be K + -selective and non-inactivating and the homologous change in two voltage-gated potassium channels retained selectivity while suppressing C-type inactivation. 46 Thus, we rationalize TOK operation via the canonical model, as follows. Inward driving force favors K + in the S1 TOK site, and thus, decreased occupancy of S2 ( Figure 8A), a state that has been associated in KcsA with an over 10fold decrease in unitary conductance, 53 and over a 10-fold decrease in open probability, 54 effects that would yield negligible inward current rapidly.…”

Selectivity filter modalities and rapid inactivation of the hERG1 channel

“…For instance, the starting [WKWKW] and [KWKWK] configurations ( Fig. 1 C and D) are to match potassium binding sites emphasized in the "water-mediated" or "soft" knock-on permeation mechanism (36)(37)(38)(39)(40). The [K0KKK] configuration ( Fig.…”

Hydration Mimicry by Membrane Ion Channels

“…Yet, new ion transport proteins, structures, functions, and mechanisms of both new and old transport proteins are discovered almost daily. This makes the field of ion channels and transporters one of the most active in molecular biology (15,35,41,68,69,70,71,72,73,74,75,76,77,78,79,80).…”