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Ionic selectivity in L-type calcium channels by electrostatics and hard-core repulsion
Journal of General Physiology, 2009
Voltage-dependent calcium channels relay electrical signals from the cell membrane to the cytoplasm by allowing extracellular Ca 2+ to flow into the cell, where Ca 2+ acts as a chemical signal . The Ca 2+ -conducting pore of these transmembrane proteins selects Ca 2+ over the much more abundant cations Na + and K + . Four amino acid residues bearing acidic side chains (the "EEEE" locus in L-type calcium channels) extend into this pore, and all cooperate in selecting Ca 2+ with high affinity for review see . Here, we compute consequences of a physical model of selectivity in which ions compete for interaction with negative structural charge and for restricted space in the EEEE locus.
Mechanisms of Permeation and Selectivity in Calcium Channels
Biophysical Journal, 2001
The mechanisms underlying ion transport and selectivity in calcium channels are examined using electrostatic calculations and Brownian dynamics simulations. We model the channel as a rigid structure with fixed charges in the walls, representing glutamate residues thought to be responsible for ion selectivity. Potential energy profiles obtained from multi-ion electrostatic calculations provide insights into ion permeation and many other observed features of L-type calcium channels. These qualitative explanations are confirmed by the results of Brownian dynamics simulations, which closely reproduce several experimental observations. These include the current-voltage curves, current-concentration relationship, block of monovalent currents by divalent ions, the anomalous mole fraction effect between sodium and calcium ions, attenuation of calcium current by external sodium ions, and the effects of mutating glutamate residues in the amino acid sequence.
Selectivity sequences in a model calcium channel: role of electrostatic field strength
European Biophysics Journal, 2011
The energetics that give rise to selectivity sequences of ionic binding selectivity of Li + , Na + , K + , Rb + , and Cs + in a model of a calcium channel are considered. This work generalizes Eisenman's classical treatment (Biophys. J. 2 (Suppl. 2), 259) by including multiple, mobile binding site oxygens that coordinate many permeating ions (all modeled as charged, hard spheres). The selectivity filter of the model calcium channel allows the carboxyl terminal groups of glutamate and aspartate side chains to directly interact with and coordinate the permeating ions. Ion dehydration effects are represented with a Born energy between the dielectric coefficients of the selectivity filter and the bath. High oxygen concentration creates a high field strength site that prefers small ions, as in Eisenman's model. On the other hand, a low filter dielectric constant also creates a high field strength site, but this site prefers large ions, contrary to Eisenman's model. These results indicate that field strength does not have a unique effect on ionic binding selectivity sequences once entropic, electrostatic, and dehydration forces are included in the model. Thus, Eisenman's classical relationship between field strength and selectivity sequences must be supplemented with additional information in selectivity filters like the calcium channel that have amino acid side chains mixing with ions to make a crowded permeation pathway.
Journal of Physical Chemistry B, 2001
Our earlier simulation (J. Phys. Chem. B 2000, 104, 8903) of the selectivity of a model calcium channel, where all ions were assumed to have the same diameter and the channel dimensions were fixed, is extended to allow for different ionic size and variable channel size. We find that for equal valence, the channel selects cations of the smallest size. If higher valence cations are present, the channel selects cations with the highest valence, and as a result, Ca ++ will replace monovalent cations. This replacement is more efficient for larger monovalent cations than for smaller ones. This is what would be expected on the basis of the charge/space competition mechanism that has been postulated earlier. Of course, if the size ratio is very large, size might be selected over valence. In addition, we consider the effect of the channel diameter and find that narrow channels are less Ca ++ selective. This suggests that the recent theory of Nonner et al. (Biophys. J. 2000(Biophys. J. , 79, 1976) is most useful for wide, but still microscopic, channels.
Journal of Membrane Biology, 2010
We explored the ability of a two-site, threebarrier (2S3B) Eyring model to describe recently reported data on current flow through open Ca V 3.1 T-type calcium channels, varying Ca 2? and Ba 2? over a wide range (100 nM-110 mM) while recording whole-cell currents over a wide voltage range (-150 mV to ?100 mV) from channels stably expressed in HEK 293 cells. Effects on permeation were isolated using instantaneous currentvoltage relationships (IIV) after strong, brief depolarizations to activate channels with minimal inactivation. Most experimental results were reproduced by a 2S3B model. The model described the IIV relationships, apparent affinities for permeation and block for Ca 2? and Ba 2? , and shifts in reversal potential between Ca 2? and Ba 2?. The fit to block by 1 mM Mg 2þ i was reasonable, but block by Mg 2þ o was described less well. Surprisingly, fits were comparable with strong ion-ion repulsion, with no repulsion, or with intermediate values. With weak repulsion, there was a single high-affinity site, with a low-affinity site near the cytoplasmic side of the pore. With strong repulsion, the net charge of ions in the pore was near ?2 over a relatively wide range of concentration and voltage, suggesting a knockoff mechanism. With strong repulsion, Ba 2? preferred the inner site, while Ca 2? preferred the outer site, potentially explaining faster entry of Ni 2? and other pore blockers when Ba 2? is the charge carrier. Keywords Eyring model Á Rate theory Á Channel block Á Chord conductance Á Ion selectivity Á Patch clamp Á Reversal potential Electronic supplementary material The online version of this article (
Computer Simulation Studies of the Selectivity and Conductance of A Model Calcium Channel
Some results of our simulation studies of selectivity and conductance of calcium ion channels in membranes are reported. Ion channels regulate many important physiological functions. To simulate the system we use periodic boundary conditions. We have noted previously that the use of such boundary conditions does not affect our results in any significant manner. The structure of the filter of a calcium channel is reasonably well understood. An important structural element is the set of four glutamate residues that are attached to the wall of the channel. Since the glutamates are long and flexible, Nonner and Eisenberg et al. have suggested that these glutamates can be modeled by eight half-charged oxygen ions that represent the carboxylates at the end of each residue. These oxygen ions are confined to the filter but otherwise are mobile. We have used this model in simulations of the selectivity and conductance of this model calcium channel filter. Our studies confirm that this model behaves as a calcium filter; calcium ions will drive sodium ions from the filter. In many of our simulations, explicit water molecules are used with water molecules in both the filter and bath.
Pore waters regulate ion permeation in a calcium release-activated calcium channel
Proceedings of the National Academy of Sciences, 2013
The recent crystal structure of Orai, the pore unit of a calcium release-activated calcium (CRAC) channel, is used as the starting point for molecular dynamics and free-energy calculations designed to probe this channel's conduction properties. In free molecular dynamics simulations, cations localize preferentially at the extracellular channel entrance near the ring of Glu residues identified in the crystal structure, whereas anions localize in the basic intracellular half of the pore. To begin to understand ion permeation, the potential of mean force (PMF) was calculated for displacing a single Na + ion along the pore of the CRAC channel. The computed PMF indicates that the central hydrophobic region provides the major hindrance for ion diffusion along the permeation pathway, thereby illustrating the nonconducting nature of the crystal structure conformation. Strikingly, further PMF calculations demonstrate that the mutation V174A decreases the free energy barrier for conduction, rendering the channel effectively open. This seemingly dramatic effect of mutating a nonpolar residue for a smaller nonpolar residue in the pore hydrophobic region suggests an important role for the latter in conduction. Indeed, our computations show that even without significant channel-gating motions, a subtle change in the number of pore waters is sufficient to reshape the local electrostatic field and modulate the energetics of conduction, a result that rationalizes recent experimental findings. The present work suggests the activation mechanism for the wild-type CRAC channel is likely regulated by the number of pore waters and hence pore hydration governs the conductance. store-operated calcium entry | computer simulation C alcium release-activated calcium (CRAC) channels in the plasma membrane are integral membrane proteins that play a central role in cellular signaling by generating the sustained influx of calcium (1-3). Immune response in cells consists typically of a fall in Ca 2+ content within the endoplasmic reticulum (ER), followed by the opening of the store-operated CRAC channels that leads to the sustained increase in intracellular Ca 2+ concentrations (4). Despite about two decades of research, the molecular components underlying this process of store-operated calcium entry (SOCE) have been unknown until recently, when the stromal interaction molecule (STIM) was determined to be the ER Ca 2+ sensor (5, 6) that activates the channel in response to the depletion of intracellular calcium store content. Later, the Orai protein was identified as the pore subunit of the channel (7-9). CRAC regulator 2A was then discovered to reinforce the binding of the two aforementioned key components at elevated Ca 2+ levels to promote the SOCE, and to inhibit the influx at low Ca 2+ levels by dissociating from the complex (10). The newly published crystal structure of the CRAC channel pore subunit, Orai, from Drosophila melanogaster at 3.35 Å resolution, provides groundbreaking insights into a molecular architecture that is distinct from that of all of the other ion channels (11). These new experiments hold promise to generate insights into the peculiar molecular mechanisms underlying the function of CRAC channels (12).
The Anomalous Mole Fraction Effect in Calcium Channels: A Measure of Preferential Selectivity
Biophysical Journal, 2008
The cause of the anomalous mole fraction effect (AMFE) in calcium-selective ion channels is studied. An AMFE occurs when the conductance through a channel is lower in a mixture of salts than in the pure salts at the same concentration. The textbook interpretation of the AMFE is that multiple ions move through the pore in coordinated, single-file motion. Instead of this, we find that at its most basic level an AMFE reflects a channel's preferential binding selectivity for one ion species over another. The AMFE is explained by considering the charged and uncharged regions of the pore as electrical resistors in series: the AMFE is produced by these regions of high and low ion concentration changing differently with mole fraction due to the preferential ion selectivity. This is demonstrated with simulations of a model L-type calcium channel and a mathematical analysis of a simplistic point-charge model. The particle simulations reproduce the experimental data of two L-type channel AMFEs. Conditions under which an AMFE may be found experimentally are discussed. The resistors-in-series model provides a fundamentally different explanation of the AMFE than the traditional theory and does not require single filing, multiple occupancy, or momentum-correlated ion motion.
Journal of General Physiology, 1986
We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by rep...
Self-organized Models of Selectivity in Ca and Na Channels
Biophysical Journal, 2009
The role of flexibility in the selectivity of calcium channels is studied using a simple model with two parameters that accounts for the selectivity of calcium (and sodium) channels in many ionic solutions of different compositions and concentrations using two parameters with unchanging values. We compare the distribution of side chains (oxygens) and cations (Na + and Ca 2+ ) and integrated quantities. We compare the occupancies of cations Ca 2+ /Na + and linearized conductance of Na + . The distributions show a strong dependence on the locations of fixed side chains and the flexibility of the side chains. Holding the side chains fixed at certain predetermined locations in the selectivity filter distorts the distribution of Ca 2+ and Na + in the selectivity filter. However, integrated quantities-occupancy and normalized conductance-are much less sensitive. Our results show that some flexibility of side chains is necessary to avoid obstruction of the ionic pathway by oxygen ions in 'unfortunate' fixed positions. When oxygen ions are mobile, they adjust 'automatically' and move 'out of the way', so they can accommodate the permeable cations in the selectivity filter. Structure is the computed consequence of the forces in this model. The structures are self-organized, at their free energy minimum. The relationship of ions and side chains varies with an ionic solution. Monte Carlo simulations are particularly well suited to compute induced-fit, self-organized structures because the simulations yield an ensemble of structures near their free energy minimum. The exact location and mobility of oxygen ions has little effect on the selectivity behavior of calcium channels. Seemingly, nature has chosen a robust mechanism to control selectivity in calcium channels: the first-order determinant of selectivity is the density of charge in the selectivity filter. The density is determined by filter volume along with the charge and excluded volume of structural ions confined within it. Flexibility seems a second-order determinant. These results justify our original assumption that the important factor in Ca 2+ versus Na + selectivity is the density of oxygen ions in the selectivity filter along with (charge) polarization (i.e. dielectric properties). The assumption of maximum mobility of oxygens seems to be an excellent approximate working hypothesis in the absence of exact structural information. These conclusions, of course, apply to what we study here. Flexibility and fine structural details may have an important role in other properties of calcium channels that are not studied in this paper. They surely have important roles in other channels, enzymes, and proteins.