Probing Ion Binding in the Selectivity Filter of the KcsA Potassium Channel.
J Am Chem Soc
; 141(18): 7391-7398, 2019 05 08.
Article
em En
| MEDLINE
| ID: mdl-30973010
ABSTRACT
In potassium (K+) channels, permeation, selectivity, and gating at the selectivity filter are all governed by the thermodynamics and kinetics of the ion-protein interactions. Specific contacts between the carbonyl groups from the Thr-Val-Gly-Tyr-Gly signature filter sequence and the permeant ions generate four equidistant K+ binding sites, thereby defining the high ion selectivity and controlling the transport rate of K+ channels. Here, we used 15N-labeled ammonium (15NH4+) as a proxy for K+ to study ion interaction with the selectivity filter of the prototypical full-length K+ channel KcsA by solution state NMR spectroscopy in order to obtain detailed insights into the physicochemical basis of K+ gating. We found that in the closed inactive state of KcsA (at pH 7) four K+ binding sites are occupied over a wide range of 15NH4+ concentrations, while in intermediate closed-open conformations (at pH â¼6) the number and occupancy of K+ binding sites are reduced to two. However, in the presence of the scorpion toxin agitoxin II a total loss of 15NH4+ binding is observed. 15NH4+ titration studies allowed us to determine the dissociation constants of the four binding sites with values around 10 mM in the closed state of KcsA. Moreover, kinetic NMR experiments measured in the steady state equilibrium detected an off- and on-rate for 15NH4+ of ca. 102 s-1 and 103 s-1 between KcsA-bound 15NH4+ and the bulk. These findings reveal both the thermodynamics and kinetics of the ion binding sites and thus contribute to our understanding of the action of K+ channels.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
Canais de Potássio
/
Compostos de Amônio
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
J Am Chem Soc
Ano de publicação:
2019
Tipo de documento:
Article