KcsA Ion Channel Research Articles

QM/MM Modeling of Ba 2+ Blockades in Potassium ion Channels

The robust selectivity of potassium channels for K+ over Na+ ions is a major component of the regulation of intracellular K+ concentrations. The selectivity for K+ was quantified through experiments measuring the Na+ and K+ dependence on Ba2+-blockades,(1) indicating that K+ has greater permeability by at least 150 fold. In thermodynamic terms, the relative binding free energy of Na+ to the pore must be at least 3 kcal/mol less favorable than K+. Na+ vs K+ free energy perturbation (FEP) simulations are consistent with this, although no simulations to date have modeled the actual Ba2+ blockade experiment. We have used MD simulations to calculate the relative binding energies of Na+ and K+ in the KcsA ion channel when the S4 site is occupied by Ba2+. As Ba2+ is a strongly polarizing ion, we have used QM/MM FEP calculations using CHARMM interfaced to the deMon DFT code (2), as well as the polarizable Drude force field to correctly model the Ba2+-filter interactions, with the aim of better interpreting the original selectivity experiments.1. J. Neyton, C. Miller, J. Gen. Physiol. 92: 549-567.2. B. Lev et al., J. Comp. Chem., 31: 1015-1023.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Semi-quantitative model of the gating of KcsA ion channel. 2. Dynamic self-organization model of the gating

The aim of this series of papers is to develop the semi-quantitative theory of the gating of KcsA channel. Methods. For this purpose available structural and electrophysiological data and the results of molecular dynamics simulations were used in the context of the concept of dynamical self-organization. In the second paper we describe the principles of dynamic self-organization and develop the theory of KcsA channel gating based on this concept. Conclusions. Present work is the first successful attempt of combining the structure and dynamics of real protein and the general concept of dynamic self-organization.

Coarse Point Charge Models For Proteins From Smoothed Molecular Electrostatic Potentials

To generate coarse electrostatic models of proteins, we developed an original approach to hierarchically locate maxima and minima in smoothed molecular electrostatic potentials. A charge-fitting program was used to assign charges to the so-obtained reduced representations. Templates are defined to easily generate coarse point charge models for protein structures, in the particular cases of the Amber99 and Gromos43A1 force fields. Applications to four small peptides and to the ion channel KcsA are presented. Electrostatic potential values generated by the reduced models are compared with the corresponding values obtained using the original sets of atomic charges.

Pulsed Electron−Electron Double-Resonance Determination of Spin-Label Distances and Orientations on the Tetrameric Potassium Ion Channel KcsA

Pulsed electron-electron double-resonance (PELDOR) measurements are presented from the potassium ion channel KcsA both solubilized in detergent and reconstituted in lipids. Site-directed spin-labeling using (1-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate was performed with a R64C mutant of the protein. The orientations of the spin-labels in the tetramer were determined by PELDOR experiments performed at two magnetic field strengths (0.3 T/X-band and 1.2 T/Q-band) and variable probe frequency. Quantitative simulation of the PELDOR data supports a strongly restricted nitroxide, oriented at an angle of 65 degrees relative to the central channel axis. In general, poorer quality PELDOR data were obtained from membrane-reconstituted preparations compared to soluble proteins or detergent-solubilized samples. One reason for this is the reduced transverse spin relaxation time T(2) of nitroxides due to crowding of tetramers within the membrane that occurs even at low protein to lipid ratios. This reduced T(2) can be overcome by reconstituting mixtures of unlabeled and labeled proteins, yielding high-quality PELDOR data. Identical PELDOR oscillation frequencies and their dependencies on the probe frequency were observed in the detergent and membrane-reconstituted preparations, indicating that the position and orientation of the spin-labels are the same in both environments.

Semi-quantitative model of the gating of KcsA ion channel. 1. Geometry and energetics of the gating

The aim of this series of papers is to develop the semi-quantitative theory of the gating of KcsA channel. For this purpose the available structural and electrophysiological data and the results of molecular dynamics simulations were used in the context of the concept of dynamical self-organization. In the first paper we describe the simplified model of the geometry and energetics of the gating process. This work is the first successful attempt of combining the structure and dynamics of a real protein and the general concept of dynamic self-organization.

Steered Molecular Dynamics Studies of the Potential of Mean Force of a Na+ or K+ Ion in a Cyclic Peptide Nanotube

Potential of mean force (PMF) profiles of a single Na+ or K+ ion passing through a cyclic peptide nanotube, cyclo[-(D-Ala-Glu-D-Ala-Gln)2-], in water are calculated to provide insight into ion transport and to understand the conductance difference between these two ions. The PMF profiles are obtained by performing steered molecular dynamics (SMD) simulations that are based on the Jarzynski equality. The computed PMF profiles for both ions show barriers of around 2.4 kcal/mol at the channel entrances and exits and energy wells in the middle of the tube. The energy barriers, so-called dielectric energy barriers, arise due to the desolvation of water molecules when ions move across the nanotube, and the energy wells appear as a result of attractive interactions between the cations and negatively charged carbonyl oxygens on the backbone of the tube. We find more and deeper energy wells in the PMF profile for Na+ than for K+, which suggests that Na+ ions have a longer residence time inside the nanotube and that permeation of Na+ ions is reduced compared to K+ ions. Calculations of the radial distribution functions (RDF) between the ions and oxygens in the water molecules and in carbonyl groups on the tube and an investigation of the orientations of the carbonyl groups show that, in contrast with the dynamic carbonyl groups observed in the selectivity filter of the KcsA ion channel, the carbonyl groups in the cyclic peptide nanotube are relatively rigid, with only slight reorientation of the carbonyl groups as the cations pass through. The rigidity of the carbonyl groups in the cyclic peptide nanotube can be attributed to their role in hydrogen bonding, which is responsible for the tube structure. Comparison of the PMF profiles with the electrostatic energy profiles calculated from the Poisson-Boltzmann (PB) equation, a dielectric continuum model, reveals that the dielectric continuum model breaks down in the confined region within the tube that governs ion transport.

Barrier-less knock-on conduction in ion channels: peculiarity or general mechanism?

Abstract A mechanism of barrier-less knock-on conduction is postulated for the best-studied KcsA ion channel. The conduction proceeds as follows: an ion enters the channel, which already contains two ions and “push” them along the pore. As a result, the state of triple occupancy forms. Eventually, the ion from the opposite side escapes to solution and the state of double occupancy is restored. The energies of the states with double and triple occupancies are almost the same, which makes alternation of these states possible. However, it is not known whether this mechanism is universal or specific for a narrow class of KcsA-like channels, which can accommodate just two or three ions in the selectivity filter. In the present work we develop a simplified model of the ion channel selectivity filter, which allows us to study the conduction in a broad range of ion occupancies using the Brownian dynamics. We show that knock-on barrier-less conduction occurs when the energies of adjacent equilibrium occupancy states are the same, regardless of the exact number of ions accommodated in the channel. It is shown that the maximal possible current flows through the channel in the regime of knock-on conduction. This allows us to conclude that knock-on barrier-less conduction can be considered as a universal mechanism of ions’ translocation in the ion channels with multiple occupancy.

A permeation theory for single-file ion channels: Concerted-association/dissociation

Motivated by atomistic simulations and x-ray analysis of the KcsA ion channel, a recent permeation model [P. H. Nelson, J. Chem. Phys. 117, 11396 (2002)] is modified to include association/dissociation via concerted motion of all n ions in the selectivity filter. The generalized kinetic model is consistent with several microscopic mechanisms, suggesting that experimentally determined parameters represent an ensemble average over all possible permeation mechanisms.

Oxygen Permeation Profile in Lipid Membranes: Comparison with Transmembrane Polarity Profile

Permeation of oxygen into membranes is relevant not only to physiological function, but also to depth determinations in membranes by site-directed spin labeling. Spin-lattice ( T 1) relaxation enhancements by air or molecular oxygen were determined for phosphatidylcholines spin labeled at positions ( n = 4–14, 16) of the sn-2 chain in fluid membranes of dimyristoyl phosphatidylcholine, by using nonlinear continuous-wave electron paramagnetic resonance (EPR). Both progressive saturation and out-of-phase continuous-wave EPR measurements yield similar oxygen permeation profiles. With pure oxygen, the T 2-relaxation enhancements determined from homogeneous linewidths of the linear EPR spectra are equal to the T 1-relaxation enhancements determined by nonlinear EPR. This confirms that both relaxation enhancements occur by Heisenberg exchange, which requires direct contact between oxygen and spin label. Oxygen concentrates in the hydrophobic interior of phospholipid bilayer membranes with a sigmoidal permeation profile that is the inverse of the polarity profile established earlier for these spin-labeled lipids. The shape of the oxygen permeation profile in fluid lipid membranes is controlled partly by the penetration of water, via the transmembrane polarity profile. At the protein interface of the KcsA ion channel, the oxygen profile is more diffuse than that in fluid lipid bilayers.