Real time dynamics of Gating-Related conformational changes in CorA.

CorA, a divalent-selective channel in the metal ion transport superfamily, is the major Mg2+-influx pathway in prokaryotes. CorA structures in closed (Mg2+-bound), and open (Mg2+-free) states, together with functional data showed that Mg2+-influx inhibits further Mg2+-uptake completing a regulatory feedback loop. While the closed state structure is a symmetric pentamer, the open state displayed unexpected asymmetric architectures. Using high-speed atomic force microscopy (HS-AFM), we explored the Mg2+-dependent gating transition of single CorA channels: HS-AFM movies during Mg2+-depletion experiments revealed the channel's transition from a stable Mg2+-bound state over a highly mobile and dynamic state with fluctuating subunits to asymmetric structures with varying degree of protrusion heights from the membrane. Our data shows that at Mg2+-concentration below Kd, CorA adopts a dynamic (putatively open) state of multiple conformations that imply structural rearrangements through hinge-bending in TM1. We discuss how these structural dynamics define the functional behavior of this ligand-dependent channel.

Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel.

Desensitization in pentameric ligand-gated ion channels plays an important role in regulating neuronal excitability. Here, we show that docosahexaenoic acid (DHA), a key ω-3 polyunsaturated fatty acid in synaptic membranes, enhances the agonist-induced transition to the desensitized state in the prokaryotic channel GLIC. We determined a 3.25 Å crystal structure of the GLIC-DHA complex in a potentially desensitized conformation. The DHA molecule is bound at the channel-periphery near the M4 helix and exerts a long-range allosteric effect on the pore across domain-interfaces. In this previously unobserved conformation, the extracellular-half of the pore-lining M2 is splayed open, reminiscent of the open conformation, while the intracellular-half is constricted, leading to a loss of both water and permeant ions. These findings, in combination with spin-labeling/EPR spectroscopic measurements in reconstituted-membranes, provide novel mechanistic details of desensitization in pentameric channels.

A chimeric prokaryotic pentameric ligand-gated channel reveals distinct pathways of activation.

Recent high resolution structures of several pentameric ligand-gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron-electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand-gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand-gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.

Stochastic-shielding approximation of Markov chains and its application to efficiently simulate random ion-channel gating.

Markov chains provide realistic models of numerous stochastic processes in nature. We demonstrate that in any Markov chain, the change in occupation number in state A is correlated to the change in occupation number in state B if and only if A and B are directly connected. This implies that if we are only interested in state A, fluctuations in B may be replaced with their mean if state B is not directly connected to A, which shortens computing time considerably. We show the accuracy and efficacy of our approximation theoretically and in simulations of stochastic ion-channel gating in neurons.

AFM and STM characterization of thiol and thiophene functionalized SWNTs: pitfalls in the use of chemical markers to determine the extent of sidewall functionalization in SWNTs.

Thiol- and thiophene-functionalized SWNTs prepared via the reaction of a substituted amine with fluoronanotubes show similar levels of sidewall functionalization, however, the use of Au nanoparticles as chemical markers for AFM gives misleading results for substituent distribution since STM shows the thiol substituents grouped in bands while the thiophene substituents uniformly distributed along the SWNTs.