Review

The structure of ion channels in membranes of excitable cells

Carbon nanotube (CNT) based transmembrane porins have attracted a lot of recent interest due to their excellent transport properties for water, ions and biomolecules. In experiments, CNTs with a length of about 10 nm can penetrate and form stable transmembrane channels across lipid membranes, while in sharp contrast, similar 10 nm long pristine CNTs were found parallelly clamped inside the hydrophobic core of a lipid bilayer in simulations. This apparent paradox has motivated the present study, where we performed all-atom molecular dynamics simulations to show that lipid molecules initially coated on the CNTs play an essential role in the formation and stabilization of the CNT porins. We demonstrate that, as a lipid coated CNT is inserted into the membrane, lipid molecules arrange themselves into a bicelle on the CNT outside the membrane, providing mechanical support to stabilize the transmembrane configuration of the CNT porin. Further analysis shows that the lipid coating density and end-functionalization strongly influence the CNT insertion into a lipid membrane. These findings shed light on the mechanism of formation and stabilization of CNT porins and provide a theoretical basis to design related transmembrane transport systems.

The overlapping of the electric double layer (EDL) in a nanochannel yields many interesting and significant electrokinetic phenomena such as ionic current rectification (ICR), which occurs only at a relatively low bulk salt concentration (∼1 mM) where the EDL thickness is comparable to the nanochannel size. In an attempt to raise this concentration to higher levels and the ICR performance improved appreciably, a branched nanochannel filled with polyelectrolytes (PEs) is proposed in this study. We show that these objectives can be achieved by choosing appropriate PE. For example, if the stem side of an anodic aluminun oxide nanochannel is filled with polystyrene sulfonate (PSS) an ICR ratio up to 850 can be obtained at 1 mM, which was not reported in previous studies. Taking account of the effect of electroosmotic flow, the underlying mechanisms of the ICR phenomena observed are discussed and the influences of the solution pH, the bulk salt concentration, and how the region(s) of a nanochannel is filled with PE examined. We show that the ICR behavior of a branched nanochannel can be modulated satisfactorily by filling highly charged PE and the solution pH.

Connexin 26 (Cx26), a protein involved in gap junctional intercellular communication, has an essential function during organ and tissue development. Its deregulation, in part due to inherent mutations, is associated with pathological conditions including congenital deafness. Regulation of Cx26 protein level is critical for its function but the molecular mechanisms involved are partially understood. This study identifies dynamin 2 (Dyn2) as a Cx26 interactor in yeast and mammalian cells. Deletion studies revealed that Cx26–Dyn2 interaction involves the C-terminus of Cx26 and the GTPase effector domain of Dyn2, which is of particular importance for the regulation of the endocytic pathway. Dyn2 inhibition using siRNA or dynasore resulted in reduced Cx26 degradation at the plasma membrane and this was associated with change in gap junctional intercellular communication (GJIC). Furthermore, we demonstrate that Dyn2 regulates Cx26 endocytosis and ubiquitination. These results establish Dyn2 as a Cx26 partner in the regulation of GJIC.

Connexin gap junctions comprise assembled channels penetrating two plasma membranes for which gating regulation is associated with a variety of factors, including voltage, pH, Ca²⁺, and phosphorylation. Functional studies have established that various parts of the connexin peptides are related to channel closure and electrophysiology studies have provided several working models for channel gating. The corresponding structural models supporting these findings, however, are not sufficient because only small numbers of closed connexin structures have been reported. To fully understand the gating mechanisms, the channels should be visualized in both the open and closed states. Electron crystallography and X-ray crystallography studies recently revealed three-dimensional structures of connexin channels in a couple of states in which the main difference is the conformation of the N-terminal domain, which have helped to clarify the structure in regard to channel closure. Here the closure models for connexin gap junction channels inferred from structural and functional studies are described in the context of each domain of the connexin protein associated with gating modulation.

A major challenge in biology is to understand how molecular processes determine phenotypic features. We address this fundamental problem in a class of model systems by developing a general mathematical framework that allows the calculation of mesoscopic properties from the knowledge of microscopic Markovian transition probabilities. We show how exact analytic formulae for the first and second moments of resident time distributions in mesostates can be derived from microscopic resident times and transition probabilities even for systems with a large number of microstates. We apply our formalism to models of the inositol trisphosphate receptor, which plays a key role in generating calcium signals triggering a wide variety of cellular responses. We demonstrate how experimentally accessible quantities, such as opening and closing times and the coefficient of variation of inter-spike intervals, and other, more elaborated, quantities can be analytically calculated from the underlying microscopic Markovian dynamics. A virtue of our approach is that we do not need to follow the detailed time evolution of the whole system, as we derive the relevant properties of its steady state without having to take into account the often extremely complicated transient features. We emphasize that our formulae fully agree with results obtained by stochastic simulations and approaches based on a full determination of the microscopic system's time evolution. We also illustrate how experiments can be devised to discriminate between alternative molecular models of the inositol trisphosphate receptor. The developed approach is applicable to any system described by a Markov process and, owing to the analytic nature of the resulting formulae, provides an easy way to characterize also rare events that are of particular importance to understand the intermittency properties of complex dynamic systems.

Molecular simulations are an invaluable tool for understanding membrane proteins. Improvements to both hardware and simulation methods have allowed access to physiologically relevant timescales and have permitted the simulation of large multimeric complexes. This, coupled to the recent expansion in membrane protein structures, provides a means to elucidate the relationship between protein structure and function. In this review, we discuss the progress in using simulations to understand the complex processes that occur at the boundary of a cell, ranging from the transport of solutes and the interactions of ligands with ion channels to the conformational rearrangements required for gating of channels and the signaling by membrane-associated complexes.