Biomedical subjects

Dirk Gillespie

Publications and source records attributed to Dirk Gillespie.

11 recordsLinked to original sources

PubMed2006

Ca2+ selectivity of a chemically modified OmpF with reduced pore volume.

We studied an E. coli OmpF mutant (LECE) containing both an EEEE-like locus, typical of Ca(2+) channels, and an accessible and reactive cysteine. After chemical modification with the cysteine-specific, negatively charged (-1e) reagents MTSES or glutathione, this LECE mutant was tested for Ca(2+) versus alkali metal selectivity. Selectivity was measured by conductance and zero-current potential. Conductance measurements showed that glutathione-modified LECE had reduced conductance at Ca(2+) mole fractions <10(-3). MTSES-modified LECE did not. Apparently, the LECE protein is (somehow) a better Ca(2+) chelator after modification with the larger glutathione. Zero-current potential measurements revealed a Ca(2+) versus monovalent cation selectivity that was highest in the presence of Li(+) and lowest in the presence of Cs(+). Our data clearly show that after the binding of Ca(2+) the LECE pore (even with the bulky glutathione present) is spacious enough to allow monovalent cations to pass. Theoretical computations based on density functional theory combined with Poisson-Nernst-Planck theory and a reduced pore model suggest a functional separation of ionic pathways in the pore, one that is specific for small and highly charged ions, and one that accepts preferentially large ions, such as Cs(+).

Calcium↗

PubMed2006

The effect of protein dielectric coefficient on the ionic selectivity of a calcium channel.

Calcium-selective ion channels are known to have carboxylate-rich selectivity filters, a common motif that is primarily responsible for their high Ca(2+) affinity. Different Ca(2+) affinities ranging from micromolar (the L-type Ca channel) to millimolar (the ryanodine receptor channel) are closely related to the different physiological functions of these channels. To understand the physical mechanism for this range of affinities given similar amino acids in their selectivity filters, we use grand canonical Monte Carlo simulations to assess the binding of monovalent and divalent ions in the selectivity filter of a model Ca channel. We use a reduced model where the electolyte is modeled by hard-sphere ions embedded in a continuum dielectric solvent, while the interior of protein surrounding the channel is allowed to have a dielectric coefficient different from that of the electrolyte. The induced charges that appear on the protein/lumen interface are calculated by the induced charge computation method [Boda et al., Phys. Rev. E 69, 046702 (2004)]. It is shown that decreasing the dielectric coefficient of the protein attracts more cations into the pore because the protein's carboxyl groups induce negative charges on the dielectric boundary. As the density of the hard-sphere ions increases in the filter, Ca(2+) is absorbed into the filter with higher probability than Na(+) because Ca(2+) provides twice the charge to neutralize the negative charge of the pore (both structural carboxylate oxygens and induced charges) than Na(+) while occupying about the same space (the charge/space competition mechanism). As a result, Ca(2+) affinity is improved an order of magnitude by decreasing the protein dielectric coefficient from 80 to 5. Our results indicate that adjusting the dielectric properties of the protein surrounding the permeation pathway is a possible way for evolution to regulate the Ca(2+) affinity of the common four-carboxylate motif.

Calcium↗

PubMed2005

Physics of size selectivity.

We demonstrate that two mechanisms used by biological ion channels to select particles by size are driven by entropy. With uncharged particles in an infinite cylinder, we show that a channel that attracts particles is small-particle selective and that a channel that repels water from the wall is large-particle selective. Comparing against the extensive density-functional theory calculations of our model, we find that the main physics can be understood with surprisingly simple bulk models that neglect the confining geometry of the channel completely.

Algorithms↗

PubMed2005

Two rings of negative charges in the cytosolic vestibule of type-1 ryanodine receptor modulate ion fluxes.

The tetrameric ryanodine receptor calcium release channels (RyRs) are cation-selective channels that have pore architecture similar to that of K+ channels. We recently identified, in close proximity to the selectivity filter motif GGGIG, a conserved lumenal DE motif that has a critical role in RyR ion permeation and selectivity. Here, we substituted three aspartate residues (D4938, D4945, D4953) with asparagine and four glutamate residues (E4942, E4948, E4952, E4955) with glutamine hypothesized to line the cytosolic vestibule of the skeletal muscle RyR (RyR1). Mutant single channel properties were determined using the planar lipid bilayer method. Two mutants (D4938N, D4945N) showed a reduced K+ ion conductance, with D4938N also exhibiting a reduced selectivity for Ca2+ compared to K+. The cytosolic location of D4938 and D4945 was confirmed using the polycation neomycin. Both D4938N and D4945N exhibited an attenuated block by neomycin to a greater extent from the cytosolic than lumenal side. By comparison, charge neutralization of lumenal loop residues (D4899Q, E4900N) eliminated the block from the lumenal but not the cytosolic side. The results suggest that, in addition to negatively charged residues on the lumenal side, rings of four negative charges formed by D4938 and D4945 in the cytosolic vestibule determine RyR ion fluxes.

Amino Acid Motifs↗

PubMed2005

(De)constructing the ryanodine receptor: modeling ion permeation and selectivity of the calcium release channel.

Biological ion channels are proteins that passively conduct ions across membranes that are otherwise impermeable to ions. Here, we present a model of ion permeation and selectivity through a single, open ryanodine receptor (RyR) ion channel. Combining recent mutation data with electrodiffusion of finite-sized ions, the model reproduces the current/voltage curves of cardiac RyR (RyR2) in KCl, LiCl, NaCl, RbCl, CsCl, CaCl(2), MgCl(2), and their mixtures over large concentrations and applied voltage ranges. It also reproduces the reduced K(+) conductances and Ca(2+) selectivity of two skeletal muscle RyR (RyR1) mutants (D4899N and E4900Q). The model suggests that the selectivity filter of RyR contains the negatively charged residue D4899 that dominates the permeation and selectivity properties and gives RyR a DDDD locus similar to the EEEE locus of the L-type calcium channel. In contrast to previously applied barrier models, the current model describes RyR as a multi-ion channel with approximately three monovalent cations in the selectivity filter at all times. Reasons for the contradicting occupancy predictions are discussed. In addition, the model predicted an anomalous mole fraction effect for Na(+)/Cs(+) mixtures, which was later verified by experiment. Combining these results, the binding selectivity of RyR appears to be driven by the same charge/space competition mechanism of other highly charged channels.

Calcium↗

PubMed2005

Probing the role of negatively charged amino acid residues in ion permeation of skeletal muscle ryanodine receptor.

Sequence comparison suggests that the ryanodine receptors (RyRs) have pore architecture similar to that of the bacterial K+ channel KcsA. The lumenal loop linking the two most C-terminal transmembrane spanning segments in the RyRs has a predicted pore helix and an amino acid motif (GGGIG) similar to the selectivity filter (TVGYG) of KcsA identified by x-ray analysis. The RyRs have many negatively charged amino acid residues in the two regions linking the GGGIG motif and predicted pore helix with the two most C-terminal transmembrane spanning segments. We tested the role of these residues by generating single-site mutants, focusing on amino acid residues conserved among the mammalian RyRs. Replacement of two acidic residues immediately after the GGGIG motif in skeletal muscle ryanodine receptor (RyR1-D4899 and -E4900) with asparagine and glutamine profoundly affected ion permeation and selectivity. By comparison, mutagenesis of aspartate and glutamate residues in the putative linker regions showed a K+ conductance and selectivity for Ca2+ compared to K+ (P(Ca)/P(K)) close to wild-type. The results show that the negatively charged carboxyl oxygens of D4899 and E4900 side chains are major determinants of RyR ion conductance and selectivity.

Amino Acid Motifs↗

PubMed2004

Relating microscopic charge movement to macroscopic currents: the Ramo-Shockley theorem applied to ion channels.

Since the discovery of gating current, electrophysiologists have studied the movement of charged groups within channel proteins by changing potential and measuring the resulting capacitive current. The relation of atomic-scale movements of charged groups to the gating current measured in an external circuit, however, is not obvious. We report here that a general solution to this problem exists in the form of the Ramo-Shockley theorem. For systems with different amounts of atomic detail, we use the theorem to calculate the gating charge produced by movements of protein charges. Even without calculation or simulation, the Ramo-Shockley theorem eliminates a class of interpretations of experimental results. The theorem may also be used at each time step of simulations to compute external current.

Algorithms↗

PubMed2004

Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca2+ channels.

The selectivity filter of the bacterial porin OmpF carries a small net charge close to -1 e and is therefore only slightly cation-selective. Calcium channels, on the other hand, contain four negatively charged glutamates, the EEEE-locus, and are among the most selective cation channels known. We aimed to turn the essentially nonselective OmpF into a Ca2+-selective channel. To that end, two additional glutamates (R42E and R132E) were introduced in the OmpF constriction zone that already contains D113 and E117. Mutant OmpF containing this DEEE-locus has a high Ca2+ over Cl- selectivity and a Na+ current with a strongly increased sensitivity to 1 mM Ca2+. The charge/space competition model, initially applied to the L-type Ca2+ channel, identifies the fixed charge and filter volume as key determinants of ion selectivity, with the precise atomic arrangement having only second-order effects. By implication, the reproduction of fixed charge and filter volume should transform two channels into channels of similar selectivity, even if the two belong to entirely different ion channel families, as is the case for OmpF and the L-type Ca2+ channel. The results presented here fit quite well in the framework of charge/space competition theory.

Calcium↗

PubMed2004

Computing induced charges in inhomogeneous dielectric media: application in a Monte Carlo simulation of complex ionic systems.

The efficient calculation of induced charges in an inhomogeneous dielectric is important in simulations and coarse-grained models in molecular biology, chemical physics, and electrochemistry. We present the induced charge computation (ICC) method for the calculation of the polarization charges based on the variational formulation of Allen et al. [Phys. Chem. Chem. Phys. 3, 4177 (2001)]. We give a different solution for their extremum condition that produces a matrix formulation. The induced charges are directly calculated by solving the linear matrix equation Ah=c, where h contains the discretized induced charge density, c depends only on the source charges-the ions moved in the simulation-and the matrix A depends on the geometry of dielectrics, which is assumed to be unchanged during the simulation. Thus, the matrix need be inverted only once at the beginning of the simulation. We verify the efficiency and accuracy of the method by means of Monte Carlo simulations for two special cases. In the simplest case, a single sharp planar dielectric boundary is present, which allows comparison with exact results calculated using the method of electrostatic images. The other special case is a particularly simple case where the matrix A is not diagonal: a slab with two parallel flat boundaries. Our results for electrolyte solutions in these special cases show that the ICC method is both accurate and efficient.

Journal Article↗

PubMed2003

Density functional theory of charged, hard-sphere fluids.

An approximate electrostatic (ES) excess free energy functional for charged, hard sphere fluids is presented. This functional is designed for systems with large density variations, but may also be applied to systems without such variations. Based on the Rosenfeld method of perturbation about a bulk (homogeneous) reference fluid [Y. Rosenfeld, J. Chem. Phys. 98, 8126 (1993)], the new ES functional replaces the reference fluid densities with a functional of the particle densities, called the RFD functional. The first-order direct correlation function (DCF) in the particle densities is computed using as input the first- and second-order DCFs in [rho(i)(x)], the inhomogeneous densities defined by the RFD functional. Because this formulation imposes no a priori constraints on the form of the RFD functional-it is valid for any choice of [rho(i)(x)]-the RFD functional may be chosen (1) so that the input DCFs (that is, DCFs in [rho(i)(x)]) may be approximated and (2) so the combination of [rho(i)(x)] and input DCFs yields a good estimate of the first-order DCF in the particle densities. In this way, the general problem of finding the excess free energy functional has been replaced by the specific problem of choosing a RFD functional. We present a particular RFD functional that, together with bulk formulations for the input DCFs, accurately reproduces the results of Monte Carlo simulations.

Biophysical Phenomena↗

PubMed2002

Physical descriptions of experimental selectivity measurements in ion channels.

Three experiments that quantify the amount of selectivity exhibited by a biological ion channel are examined with Poisson-Nernst-Planck (PNP) theory. Conductance ratios and the conductance mole fraction experiments are examined by considering a simple model ion channel for which an approximate solution to the PNP equations with Donnan boundary conditions is derived. A more general result is derived for the Goldman-Hodgkin-Katz permeability ratio. The results show that (1) the conductance ratio measures the ratio of the diffusion coefficients of the ions inside the channel, (2) the mole fraction experiment measures the difference of the excess chemical potentials of the ions inside the channel, and (3) the permeability ratio measures both diffusion coefficients and excess chemical potentials. The results are used to divide selectivity into two components: partitioning, an equilibrium measure of how well the ions enter the channel, and diffusion, a nonequilibrium measure of how well the ions move through the channel.

Cell Membrane↗