Conformational Dynamics in the Selectivity Filter of KcsA in Response to Potassium Ion Concentration

Abstract

The conformational change in the selectivity filter of KcsA as a function of ambient potassium concentration is studied with solid state NMR. This highly conserved region of the protein is known to chelate potassium ions selectively. We report solid-state NMR chemical shift fingerprints of two distinct conformations of the selectivity filter; significant changes are observed in the chemical shifts of key residues in the filter as the buffer potassium ion concentration is changed from 50 mM to 1 µM. Potassium ion titration studies reveal that the site-specific K d for K + binding at the key pore residue Val 76, is on the order of ∼7 µM and that relatively high sample hydration is necessary to observe the low K + conformer. Simultaneous detection of both conformers at low ambient potassium concentration suggests that the high K + and low K + states are in slow exchange on the NMR timescale (k ex < 500 s −1 ). The slow rate and tight binding for evacuating both inner sites simultaneously, differ from prior observations in detergents in solution, but agree well with measurements by electrophysiology, and appear to result from our use of a hydrated bilayer environment. These characteristics rule out participation of the low K + state on the timescale of ion transmission, which has been assumed to involve interchange of states where one of the inner binding sites is always occupied. On the other hand, these kinetic and thermodynamic characteristics of evacuation of the inner sites certainly could be compatible with participation in a control mechanism at low ion concentration, such as C-type inactivation, a process that is coupled to activation and involves closing of the outer mouth of the channel.

Transmembrane allosteric coupling of the gates in a potassium channel

“…Previously we reported on NMR spectra of full-length KcsA in bilayers at low [K + ], and using NMR reporters near the selectivity filter that are well dispersed spectrally, we documented a conformational change upon ion binding and an ion affinity in the low micromolar range (9). Unique chemical shift assignments obtained from the present study attest to global conformational effects of K + binding.…”

“…In separate experiments, the extracellular gate has been observed to respond directly to ambient [K + ]: at high [K + ] it exists in a conductive form, and at low K + it collapses into a nonconductive state (3). Our NMR studies suggest that the low [K + ] state and the low pH inactivated state may be similar; this conclusion is supported by the effect of the mutation E71A and the pattern of chemical shift perturbations in the selectivity filter when the ion is depleted (9,19). Meanwhile, X-ray crystallography studies suggest that mutants (E71A) unable to undergo inactivation are also unable to expel ions (26).…”

“…KcsA is a 160-residue pH-activated homotetrameric K + channel isolated from the soil bacterium Streptomyces lividans (2, 3) with high sequence homology and functional similarity to mammalian potassium channels (4). It has provided an excellent model for studies of ion-conduction by X-ray crystallography (3,5,6), electrophysiology (7,8), and NMR (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Like many potassium channels, it exhibits (4, 6, 22, 23) slow, spontaneous inactivation involving the residues near the extracellular selectivity filter subsequent to channel activation.…”

Selective exclusion and selective binding both contribute to ion selectivity in KcsA, a model potassium channel

“…Such K + concentration-dependent conformational changes were also seen in NMR studies (9)(10)(11)(12)(13)(14). Low K + concentrations leads to a nonconductive conformation of the selectivity filter in which K + binds only at the extreme S1 and S4 sites, with an average occupancy of just one K + distributed between those two sites.…”

Filter gate closure inhibits ion but not water transport through potassium channels

“…Bulk K + concentrations ≥9 mM are compatible with this view. Although osmotic water flow through the filter sweeps away ions, the experimental conditions ensure multiple visits of K + to all binding sites within the time required for inactivation (26). Thus, there is only a small probability of filter collapse, as documented by X-ray crystallography for a lone K + equilibrated in its binding pocket (5,27).…”