Structural modeling of calcium binding in the selectivity filter of the L-type calcium channel.
ABSTRACT Calcium channels play crucial physiological roles. In the absence of high-resolution structures of the channels, the mechanism of ion permeation is unknown. Here we used a method proposed in an accompanying paper (Cheng and Zhorov in Eur Biophys J, 2009) to predict possible chelation patterns of calcium ions in a structural model of the L-type calcium channel. We compared three models in which two or three calcium ions interact with the four selectivity filter glutamates and a conserved aspartate adjacent to the glutamate in repeat II. Monte Carlo energy minimizations yielded many complexes with calcium ions bound to at least two selectivity filter carboxylates. In these complexes calcium-carboxylate attractions are counterbalanced by calcium-calcium and carboxylate-carboxylate repulsions. Superposition of the complexes suggests a high degree of mobility of calcium ions and carboxylate groups of the glutamates. We used the predicted complexes to propose a permeation mechanism that involves single-file movement of calcium ions. The key feature of this mechanism is the presence of bridging glutamates that coordinate two calcium ions and enable their transitions between different chelating patterns involving four to six oxygen atoms from the channel protein. The conserved aspartate is proposed to coordinate a calcium ion incoming to the selectivity filter from the extracellular side. Glutamates in repeats III and IV, which are most distant from the repeat II aspartate, are proposed to coordinate the calcium ion that leaves the selectivity filter to the inner pore. Published experimental data and earlier proposed permeation models are discussed in view of our model.
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ABSTRACT: Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+).The Journal of General Physiology 07/2007; 129(6):477-83. · 4.73 Impact Factor
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ABSTRACT: The chromanol 293B (293B, trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman) is a lead compound of potential class III antiarrhythmics that inhibit cardiac I(Ks) potassium channels. These channels are formed by the coassembly of KCNQ1 (Kv7.1, KvLQT1) and KCNE1 subunits. Although homomeric KCNQ1 channels are the principal molecular targets, entry of KCNE1 to the channel complex enhances the chromanol block. Because closely related neuronal KCNQ2 potassium channels are insensitive to the drug, we used KCNQ1/KCNQ2 chimeras to identify the binding site of the inhibitor. We localized the putative drug receptor to the H5 selectivity filter and the S6 transmembrane segment. Single residues affecting 293B inhibition were subsequently identified through systematic exchange of amino acids that were either different in KCNQ1 and KCNQ2 or predicted by a docking model of 293B in the open and closed conformation of KCNQ1. Mutant channel proteins T312S, I337V, and F340Y displayed dramatically lowered sensitivity to chromanol block. The predicted drug binding receptor lies in the inner pore vestibule containing the lower part of the selectivity filter, and the S6 transmembrane domain also reported to be important for binding of benzodiazepines. We propose that the block of the ion permeation pathway involves hydrophobic interactions with the S6 transmembrane residues Ile337 and Phe340, and stabilization of chromanol 293B binding through electrostatic interactions of its oxygen atoms with the most internal potassium ion within the selectivity filter.Molecular Pharmacology 07/2007; 71(6):1503-11. · 4.41 Impact Factor
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ABSTRACT: A method of docking Ca(2+) ions in proteins with flexible side chains and deformable backbones is proposed. The energy was calculated with the AMBER force field, implicit solvent, and solvent exposure-dependent and distance-dependent dielectric function. Starting structures were generated with Ca(2+) coordinates and side-chain torsions sampled in 1000 A(3) cubes centered at the experimental Ca(2+) positions. The energy was Monte Carlo-minimized. The method was tested on fourteen Ca(2+)-binding sites. For twelve Ca(2+)-binding sites the root mean square (RMS) deviation of the apparent global minimum from the experimental structure was below 1.3 and 1.7 A for Ca(2+) ions and side-chain heavy atoms, respectively. Energies of multiple local minima correlate with the RMS deviations from the X-ray structures. Two Ca(2+)-binding sites at the surface of proteinase K were not predicted, because of underestimation of Ca(2+) hydration energy by the implicit-solvent method.Biophysics of Structure and Mechanism 11/2009; 39(5):825-38. · 2.44 Impact Factor