Takashi Sumikama

The Open Gate Structure of the Membrane-Embedded KcsA Potassium Channel Viewed From the Cytoplasmic Side

Crystallographic studies of channel proteins have provided insight into the molecular mechanisms of ion channels, even though these structures are obtained in the absence of the membrane and some structural portions have remained unsolved. Here we report the gating structure of the membrane-embedded KcsA potassium channel using atomic force microscopy (AFM). The activation gate of the KcsA channel is located on the intracellular side, and the cytoplasmic domain was truncated to clear the view of this location. Once opened, the individual subunits in the tetramer were resolved with the pore open at the center. Furthermore, AFM was able to capture the previously unsolved bulge helix at the entrance. A molecular dynamics simulation revealed that the bulge helices fluctuated dramatically at the open entryway. This dynamic behavior was observed as vigorous open-channel noise in the single-channel current recordings. The role of the bulge helices in the open gate structure is discussed.

Cycle flux algebra for ion and water flux through the KcsA channel single-file pore links microscopic trajectories and macroscopic observables.

In narrow pore ion channels, ions and water molecules diffuse in a single-file manner and cannot pass each other. Under such constraints, ion and water fluxes are coupled, leading to experimentally observable phenomena such as the streaming potential. Analysis of this coupled flux would provide unprecedented insights into the mechanism of permeation. In this study, ion and water permeation through the KcsA potassium channel was the focus, for which an eight-state discrete-state Markov model has been proposed based on the crystal structure, exhibiting four ion-binding sites. Random transitions on the model lead to the generation of the net flux. Here we introduced the concept of cycle flux to derive exact solutions of experimental observables from the permeation model. There are multiple cyclic paths on the model, and random transitions complete the cycles. The rate of cycle completion is called the cycle flux. The net flux is generated by a combination of cyclic paths with their own cycle flux. T.L. Hill developed a graphical method of exact solutions for the cycle flux. This method was extended to calculate one-way cycle fluxes of the KcsA channel. By assigning the stoichiometric numbers for ion and water transfer to each cycle, we established a method to calculate the water-ion coupling ratio (CR w-i) through cycle flux algebra. These calculations predicted that CR w-i would increase at low potassium concentrations. One envisions an intuitive picture of permeation as random transitions among cyclic paths, and the relative contributions of the cycle fluxes afford experimental observables.

A mesoscopic approach to understanding the mechanisms underlying the ion permeation on the discrete-state diagram

In the [Perspectives][1] series in the June 2010 issue ([Bahar, 2010][2]; [Bucher and Rothlisberger, 2010][3]; [Dror et al., 2010][4]; [Roux, 2010][5]; [Silva and Rudy, 2010][6]), a broad range of permeation events from femtoseconds to minutes in time scale and sub-angstrom to millimeter in space

Mechanism of ion permeation through a model channel: Roles of energetic and entropic contributions

Mechanism of ion permeation through an anion-doped carbon nanotube (ANT), a model of ion channel, is investigated. Using this model system, many trajectory calculations are performed to obtain the potential energy profile, in addition to the free energy profile, that enables to separate the energy and the entropic contributions, along the ion permeation. It is found that the mechanism of the transport is governed by the interplay between the energetic and the entropic forces. The rate of the ion permeation can be controlled by changing the balance between these contributions with altering, for example, the charge and/or the length of ANT, which increases the rate of the ion permeation by nearly two orders of magnitude. The dominant free energy barrier at the entrance of ANT is found to be caused by the entropy bottleneck due to the narrow phase space for the exchange of a water molecule and an incoming ion.

Capturing the Freeze-Drying Dynamics of NaCl Nanoparticles Using THz Spectroscopy

Sodium chloride (NaCl) aqueous solution becomes NaCl hydrate, NaCl·2H2O, at low temperature, which is different from potassium chloride and is a typical complex model for studying the freeze-drying process in foods and pharmaceuticals. Here, we detected unit-cell-sized NaCl particles in ice as precursor substances of NaCl·2H2O during freezing of NaCl solution by using terahertz (THz) spectroscopy. In the freezing process, Na+ and Cl- ions form two types of metastable unit-cell-sized NaCl particles on the pathway to the well-known NaCl·2H2O crystal production, which are not listed in the phase diagram of freezing of NaCl solution but have absorption peaks in THz spectra. This finding of single unit-cell-sized particles signifies the importance of studying the freeze-drying process in-depth and offers a new possibility for the development of freeze-drying technology for the manufacture of nanometer-sized particles such as ultrafine pharmaceutical powders, which more readily dissolve in water.

Origin of the Shape of Current-Voltage Curve through Nanopores: A Molecular Dynamics Study.

Ion transports through ion channels, biological nanopores, are essential for life: Living cells generate electrical signals by utilizing ion permeation through channels. The measured current-voltage (i-V) relations through most ion channels are sublinear, however, its physical meaning is still elusive. Here we calculated the i-V curves through anion-doped carbon nanotubes, a model of an ion channel, using molecular dynamics simulation. It was found the i-V curve reflects the physical origin of the rate-determining step: the i-V curve is sublinear when the permeation is entropy bottlenecked, while it is superlinear in the case of the energy bottlenecked permeation. Based on this finding, we discuss the relation between the molecular mechanism of ion permeation through the biological K+ channels and the shape of the i-V curves through them. This work also provides a clue for a novel design of nanopores that show current rectification.