TY - JOUR
T1 - Mechanism of Intracellular Block of the KcsA K+Channel by Tetrabutylammonium: Insights from X-ray Crystallography, Electrophysiology and Replica-exchange Molecular Dynamics Simulations
AU - Faraldo-Gómez, José D.
AU - Kutluay, Esin
AU - Jogini, Vishwanath
AU - Zhao, Yanxiang
AU - Heginbotham, Lise
AU - Roux, Benoît
PY - 2007/1/19
Y1 - 2007/1/19
N2 - The mechanism of intracellular blockade of the KcsA potassium channel by tetrabutylammonium (TBA) is investigated through functional, structural and computational studies. Using planar-membrane electrophysiological recordings, we characterize the binding kinetics as well as the dependence on the transmembrane voltage and the concentration of the blocker. It is found that the apparent affinity of the complex is significantly greater than that of any of the eukaryotic K+channels studied previously, and that the off-rate increases with the applied transmembrane voltage. In addition, we report a crystal structure of the KcsA-TBA complex at 2.9 Å resolution, with TBA bound inside the large hydrophobic cavity located at the center of the channel, consistent with the results of previous functional and structural studies. Of particular interest is the observation that the presence of TBA has a negligible effect on the channel structure and on the position of the potassium ions occupying the selectivity filter. Inspection of the electron density corresponding to TBA suggests that the ligand may adopt more than one conformation in the complex, though the moderate resolution of the data precludes a definitive interpretation on the basis of the crystallographic refinement methods alone. To provide a rationale for these observations, we carry out an extensive conformational sampling of an atomic model of TBA bound in the central cavity of KcsA, using the Hamiltonian replica-exchange molecular dynamics simulation method. Comparison of the simulated and experimental density maps indicates that the latter does reflect at least two distinct binding orientations of TBA. The simulations show also that the relative population of these binding modes is dependent on the ion configuration occupying the selectivity filter, thus providing a clue to the nature of the voltage-dependence of the binding kinetics.
AB - The mechanism of intracellular blockade of the KcsA potassium channel by tetrabutylammonium (TBA) is investigated through functional, structural and computational studies. Using planar-membrane electrophysiological recordings, we characterize the binding kinetics as well as the dependence on the transmembrane voltage and the concentration of the blocker. It is found that the apparent affinity of the complex is significantly greater than that of any of the eukaryotic K+channels studied previously, and that the off-rate increases with the applied transmembrane voltage. In addition, we report a crystal structure of the KcsA-TBA complex at 2.9 Å resolution, with TBA bound inside the large hydrophobic cavity located at the center of the channel, consistent with the results of previous functional and structural studies. Of particular interest is the observation that the presence of TBA has a negligible effect on the channel structure and on the position of the potassium ions occupying the selectivity filter. Inspection of the electron density corresponding to TBA suggests that the ligand may adopt more than one conformation in the complex, though the moderate resolution of the data precludes a definitive interpretation on the basis of the crystallographic refinement methods alone. To provide a rationale for these observations, we carry out an extensive conformational sampling of an atomic model of TBA bound in the central cavity of KcsA, using the Hamiltonian replica-exchange molecular dynamics simulation method. Comparison of the simulated and experimental density maps indicates that the latter does reflect at least two distinct binding orientations of TBA. The simulations show also that the relative population of these binding modes is dependent on the ion configuration occupying the selectivity filter, thus providing a clue to the nature of the voltage-dependence of the binding kinetics.
KW - binding
KW - Cation
KW - hydrophobic
KW - molecular dynamics
KW - solvation
UR - http://www.scopus.com/inward/record.url?scp=33845663257&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2006.09.069
DO - 10.1016/j.jmb.2006.09.069
M3 - Journal article
C2 - 17070844
SN - 0022-2836
VL - 365
SP - 649
EP - 662
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -