Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli.

NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments. We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM: KCl to 3 M: KCl), (2) anion over cation selectivity (V (reversal) = +16 mV in 250 mM: KCl || 1 M: KCl), and (3) two forms of voltage-dependent gating (channel closures above ± 200 mV). Single-channel conductance decreases by 50% when HEPES concentration is increased from 100 µM: to 100 mM: in 250 mM: KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers, we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate concentration is increased from 0 mM: to 5 mM: in 250 mM: KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes are on the far side of agar-KCl bridges. A suitable baseline solution for NanC is 250 mM: KCl adjusted to pH 7.0 without buffer.

Self-organized models of selectivity in calcium channels.

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.

Analyzing the components of the free-energy landscape in a calcium selective ion channel by Widom's particle insertion method.

The selectivity filter of the L-type calcium channel works as a Ca(2+) binding site with a very large affinity for Ca(2+) versus Na(+). Ca(2+) replaces half of the Na(+) ions in the filter even when these ions are present in 1 µM and 30 mM concentrations in the bath, respectively. The energetics of this strong selectivity is analyzed in this paper. We use Widom's particle insertion method to compute the space-dependent profiles of excess chemical potential in our grand canonical Monte Carlo simulations. These profiles define the free-energy landscape for the various ions. Following Gillespie [Biophys. J. 94, 1169 (2008)], the difference of the excess chemical potentials for the two competing ions defines the advantage that one of the ions has over the other in the competition for space in the crowded selectivity filter. These advantages depend on ionic bath concentrations: the ion that is present in the bath in larger quantity (Na(+)) has the "number" advantage which is balanced by the free-energy advantage of the other ion (Ca(2+)). The excess chemical potentials are decomposed into hard sphere exclusion and electrostatic components. The electrostatic terms correspond to interactions with the mean electric field produced by ions and induced charges as well to ionic correlations beyond the mean field description. Dielectrics are needed to produce micromolar Ca(2+) versus Na(+) selectivity in the L-type channel. We study the behavior of these terms with changes in bath concentrations of ions, charges, and diameters of ions, as well as geometrical parameters such as radius of the pore and the dielectric constant of the protein. Ion selectivity in calcium binding proteins probably has a similar mechanism.

Reinterpreting the anomalous mole fraction effect: the ryanodine receptor case study.

The origin of the anomalous mole fraction effect (AMFE) in calcium channels is explored with a model of the ryanodine receptor. This model predicted and experiments verified new AMFEs in the cardiac isoform. In mole fraction experiments, conductance is measured in mixtures of ion species X and Y as their relative amounts (mole fractions) vary. This curve can have a minimum (an AMFE). The traditional interpretation of the AMFE is that multiple interacting ions move through the pore in a single file. Mole fraction curves without minima (no AMFEs) are generally interpreted as X displacing Y from the pore in a proportion larger than its bath mole fraction (preferential selectivity). We find that the AMFE is also caused by preferential selectivity of X over Y, if X and Y have similar conductances. This is a prediction applicable to any channel and provides a fundamentally different explanation of the AMFE that does not require single filing or multiple occupancy: preferential selectivity causes the resistances to current flow in the baths, channel vestibules, and selectivity filter to change differently with mole fraction, and produce the AMFE.