Study of Selectivity and Permeation in Voltage-Gated Ion Channels

Ion channels are proteins with holes down their middle that regulate access to biological cells and are responsible for an enormous range of biological functions. A substantial fraction of the drugs used in clinical medicine are targeted directly or indirectly on channels. Ion selectivity and permeation are based on simple laws of physics and chemistry. Ion channels are therefore ideal candidates for physical investigation. A reduced model generates the selectivity of voltage-gated L-type calcium channel (crystal structure not known) under a wide range of ionic conditions using only two parameters with unchanging values. The reasons behind the success of this reduced model are investigated since chemical intuition suggests that more detailed models are needed. Monte Carlo simulations are performed investigating the role of flexibility of the side chains in the selectivity of calcium channels under a wide range of ionic conditions. Results suggest that the exact location and mobility of oxygen ions have little effect on the selectivity behavior of calcium channels. The first order determinant of selectivity in calcium channels is the density of charge in the selectivity filter. Flexibility seems a second order determinant. Single channel planar lipid bilayer experiments are performed to determine the selectivity, permeation, and sialic acid specificity of a bacterial outer-membrane channel NanC of Escherichia Coli with a known crystal structure. Measurements show that NanC exhibits a large unit conductance varying with the applied voltage polarity, anion over cation selectivity, and voltage-dependent gating. Unitary conductance of NanC decreases significantly in presence of the buffer HEPES. Alternate buffers are tested. Results suggest that the sialic acid specificity of NanC should be performed in low concentration salt solutions (250 mM) without pH buffers adjusted to neutral pH 7.0. Neu5Ac (a major sialic acid) is observed to change the gating and considerably increase the ionic conductance of NanC. The effect of Neu5Ac on the unitary current through NanC saturates at higher Neu5Ac concentrations. Interestingly, Neu5Ac reduces the ionic conductance of OmpF: frequent, long closures are seen. Therefore, in E.coli sialic acid translocation is specifically facilitated by NanC, and not by the general porin OmpF.