Comment on ion transit pathways and gating in ClC chloride channels

Yin et al. presented a computational study of ion transit pathways through the bacterial chloride channel homologues. Pathways for the permeation of both chloride ions and protons were explored using a new biased Monte Carlo algorithm, TransPath. The potential and radius profiles along the various pathways provide evidence for the origin of ion selectivity. Two possible pathways for access of protons from the extracellular solution to the Glu148 gate were found: one along the edge of the entrance vestibule and one inside the protein but not far from the vestibular path. The Glu148 gate blocks the pore in the closed state. Based on the computed properties for the chloride pathway, the electrostatic potential (due to the protein and the dielectric environment) near the Glu148 residue is large and positive. This implies a large negative shift in the pKa of the glutamate residue. Yet the chloride channels and bacterial homologues exhibit extracellular pH-dependent gating. To examine the possibility of protonation of the glutamate gate, we performed continuum electrostatics calculations for the Glu148 residue with no chloride ions present, with two ions at the Sint and Scen sites, and with a third ion at a proposed binding site Sbs outside of the selectivity filter near Arg147 in the extracellular vestibule. We observed a large negative pKa shift with no chloride ions present, a shift of 0.67 unit with the two ions at Sint and Scen, and a shift of 3.33 units with the third ion present at Sbs. Recently, we found that errors were made in our calculations in the assignment of the dielectric constant in the region around the Glu148 residue, which led to an overestimation of the chloride-induced pKa shift. The membrane dielectric constant m was extended into the extracellular vestibular region. Also, a mesh size of 0.5 Å was used, which is somewhat large for the inhomogeneous environment of the Glu148 residue. This comment reports the corrected results, including testing of the mesh size, and further it examines in detail the dependence of the predicted pKa shift on the protein dielectric constant. The general conclusion doesn’t change in that it is highly likely that chloride ions in the filter and possibly nearby in the extracellular vestibule are required to allow for protonation of the Glu148 gate; the predicted magnitude of the chloride-induced shift is less than in our previously published result, however. Continuum electrostatics calculations were performed with the PBEQ module of CHARMM28. The closed bacterial ClC structure 1OTS [3] was studied in the presence of 0, 2, and 3 chloride ions located at the [Sint, Scen] and [Sint, Scen, Sbs] sites, respectively. The Sint and Scen sites are resolved in the X-ray structure, while the Sbs site is not resolved. The Sbs site was chosen over a range of z values along the chloride ion path located by our TransPath algorithm. The linearized Poisson–Boltzmann equation was solved assuming a salt concentration of 0.150 M, a 2.0-Å Stern layer, and a water-probe radius of 1.4 Å for Richard Probes molecular surface. CHARMM 27 parameters were used for atomic radii and atomic charges. The dielectric constant of the solvent was assigned as 80. The dielectric constant for the molecular interior was varied from 4 to 80. Grid focusing with different mesh sizes was studied, and the initial mesh spacing for the whole protein was assigned to be 0.5 Å. The membrane thickness was varied between 23 and 35 Å and the membrane dielectric was assigned as 2. A central cylinder of radius 33 Å and dielectric constant of the solvent was included to exclude the membrane from the vestibular region. Table I reports the dependence of the pKa shift on mesh size for the case with 2 chloride ions located at Sint and Scen. The protein dielectric constant here is taken as 20.0 while the solvent dielectric constant is 80.0. The grid dimensions are 35 Å in excess of the protein in each direction. The thickness of the membrane is 35 Å. The ionic strength is 150 mM. The dependence of the pKa on mesh size is relatively weak, changing by roughly 0.3 over the chosen range. The dependence of the shift on the thickness of the membrane is examined in Table II. The parameters are the same as for the mesh size study described above, and a mesh size of 0.25 Å was chosen. The dependence of the shift on membrane thickness is also not strong, changing