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(+).

[1]  W. Im,et al.  Ion permeation and selectivity of OmpF porin: a theoretical study based on molecular dynamics, Brownian dynamics, and continuum electrodiffusion theory. , 2002, Journal of molecular biology.

[2]  W. Stühmer,et al.  Calcium channel characteristics conferred on the sodium channel by single mutations , 1992, Nature.

[3]  G. Rummel,et al.  Crystal structures explain functional properties of two E. coli porins , 1992, Nature.

[4]  H. Bayley,et al.  Reversal of charge selectivity in transmembrane protein pores by using noncovalent molecular adapters. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[5]  R. Tsien,et al.  Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels , 1993, Nature.

[6]  Dirk Gillespie,et al.  Ion Accumulation in a Biological Calcium Channel: Effects of Solvent and Confining Pressure , 2001 .

[7]  G. Schulz The structure of bacterial outer membrane proteins. , 2002, Biochimica et biophysica acta.

[8]  W. Im,et al.  Ions and counterions in a biological channel: a molecular dynamics simulation of OmpF porin from Escherichia coli in an explicit membrane with 1 M KCl aqueous salt solution. , 2002, Journal of molecular biology.

[9]  R. Eisenberg,et al.  A physical mechanism for large-ion selectivity of ion channels , 2002 .

[10]  J. Rosenbusch,et al.  Role of charged residues at the OmpF porin channel constriction probed by mutagenesis and simulation. , 2001, Biochemistry.

[11]  Abraham Nitzan,et al.  The role of the dielectric barrier in narrow biological channels: a novel composite approach to modeling single-channel currents. , 2003, Biophysical journal.

[12]  B. Nadler,et al.  Saturation of conductance in single ion channels: the blocking effect of the near reaction field. , 2004, Physical review. E, Statistical, nonlinear, and soft matter physics.

[13]  Robert S. Eisenberg,et al.  Physical descriptions of experimental selectivity measurements in ion channels , 2002, European Biophysics Journal.

[14]  R. Tsien,et al.  Mechanism of ion permeation through calcium channels , 1984, Nature.

[15]  W. Sather,et al.  The EEEE Locus Is the Sole High-affinity Ca 2 1 Binding Structure in the Pore of a Voltage-gated Ca 2 1 Channel Block by Ca 2 1 Entering from the Intracellular Pore Entrance , 2000 .

[16]  P. Phale,et al.  Brownian dynamics simulation of ion flow through porin channels. , 1999, Journal of molecular biology.

[17]  A. Delcour Solute uptake through general porins. , 2003, Frontiers in bioscience : a journal and virtual library.

[18]  A. Nitzan,et al.  A lattice relaxation algorithm for three-dimensional Poisson-Nernst-Planck theory with application to ion transport through the gramicidin A channel. , 1999, Biophysical journal.

[19]  L Schild,et al.  On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel. , 1996, Biophysical journal.

[20]  P. Kienker,et al.  Charge selectivity of the designed uncharged peptide ion channel Ac-(LSSLLSL)3-CONH2. , 1995, Biophysical journal.

[21]  Dirk Gillespie,et al.  (De)constructing the ryanodine receptor: modeling ion permeation and selectivity of the calcium release channel. , 2005, The journal of physical chemistry. B.

[22]  Edward Moczydlowski,et al.  On the Structural Basis for Size-selective Permeation of Organic Cations through the Voltage-gated Sodium Channel , 1997, The Journal of general physiology.

[23]  Robert S. Eisenberg,et al.  Ion flow through narrow membrane channels: part II , 1992 .

[24]  Tilman Schirmer General and specific porins from bacterial outer membranes. , 1998, Journal of structural biology.

[25]  A. Warshel,et al.  Calculations of electrostatic interactions in biological systems and in solutions , 1984, Quarterly Reviews of Biophysics.

[26]  Ion channel selectivity using an electric stew. , 2000, Biophysical journal.

[27]  B. Honig,et al.  Classical electrostatics in biology and chemistry. , 1995, Science.

[28]  B. Roux,et al.  Control of ion selectivity in potassium channels by electrostatic and dynamic properties of carbonyl ligands , 2004, Nature.

[29]  N. Klugbauer,et al.  Aspartate Residues of the Glu-Glu-Asp-Asp (EEDD) Pore Locus Control Selectivity and Permeation of the T-type Ca2+Channel α1G * , 2001, The Journal of Biological Chemistry.

[30]  R. Tsien,et al.  Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore , 1986, The Journal of general physiology.

[31]  N. Saint,et al.  Structural and Functional Characterization of OmpF Porin Mutants Selected for Larger Pore Size , 1996, The Journal of Biological Chemistry.

[32]  W. Almers,et al.  Non‐selective conductance in calcium channels of frog muscle: calcium selectivity in a single‐file pore. , 1984, The Journal of physiology.

[33]  M. Saraniti,et al.  A Poisson P3M Force Field Scheme for Particle-Based Simulations of Ionic Liquids , 2004 .

[34]  W. Sather,et al.  The Eeee Locus Is the Sole High-Affinity Ca2+ Binding Structure in the Pore of a Voltage-Gated Ca2+ Channel , 2000, The Journal of general physiology.

[35]  B. Eisenberg,et al.  Ion permeation and glutamate residues linked by Poisson-Nernst-Planck theory in L-type calcium channels. , 1998, Biophysical journal.

[36]  Umberto Ravaioli,et al.  BioMOCA—a Boltzmann transport Monte Carlo model for ion channel simulation , 2005 .

[37]  B. Eisenberg,et al.  Binding and selectivity in L-type calcium channels: a mean spherical approximation. , 2000, Biophysical journal.

[38]  Shin-Ho Chung,et al.  Electrostatic basis of valence selectivity in cationic channels. , 2005, Biochimica et biophysica acta.

[39]  Dirk Gillespie,et al.  Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca2+ channels. , 2004, Biophysical journal.

[40]  H. Miedema,et al.  Chemical modification of the bacterial porin OmpF: gain of selectivity by volume reduction. , 2006, Biophysical journal.

[41]  Bob Eisenberg,et al.  Monte Carlo simulations of ion selectivity in a biological Na channel: Charge–space competition , 2002 .

[42]  Christopher Miller,et al.  Electrostatic tuning of ion conductance in potassium channels. , 2003, Biochemistry.

[43]  R. S. Eisenberg,et al.  Computing the Field in Proteins and Channels , 2010, 1009.2857.

[44]  Bob Eisenberg,et al.  Proteins, channels and crowded ions. , 2002, Biophysical chemistry.

[45]  Robert S. Eisenberg,et al.  Coupling Poisson–Nernst–Planck and density functional theory to calculate ion flux , 2002 .

[46]  Uwe Hollerbach,et al.  Dielectric boundary force and its crucial role in gramicidin. , 2003, Physical review. E, Statistical, nonlinear, and soft matter physics.

[47]  R. Tsien,et al.  Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells , 1986, The Journal of general physiology.

[48]  Ernst Bamberg,et al.  ION TRANSPORT THROUGH THE GRAMICIDIN A CHANNEL , 1976 .

[49]  Dirk Gillespie,et al.  Density functional theory of charged, hard-sphere fluids. , 2003, Physical review. E, Statistical, nonlinear, and soft matter physics.

[50]  B. Nadler,et al.  Derivation of Poisson and Nernst-Planck equations in a bath and channel from a molecular model. , 2001, Physical review. E, Statistical, nonlinear, and soft matter physics.

[51]  J. Kasianowicz,et al.  Conductance and ion selectivity of a mesoscopic protein nanopore probed with cysteine scanning mutagenesis. , 2005, Biophysical journal.