Sodium channel investigation

Researchers at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University report in Nature Communications a high-speed atomic force microscopy study of the structural dynamics of sodium ion channels in cell membranes. The findings provide insights into the mechanism behind the generation of cell-membrane action potentials.

Figure 1.
HS-AFM of the Nav channels. (a and e) Snapshots of HS-AFM movies of WT (open) and E32Q/N49K (closed) channels. (b and f) Time-averaged images of (a) and (e). (c and g) Localization AFM images of (a) and (e). (d and h) Schematic illustration of results.

The transport of ions to and from a cell is controlled by pore-forming proteins embedded in the cell membrane.  In particular, so-called voltage-gated sodium channels (VGSCs) govern the transfer of sodium (Na+) ions, and play an important role in the regulation of the membrane potential — the voltage difference between the cell’s exterior and interior.  In electrically excitable cells such as neurons and muscle cells, VGSCs participate in the generation of action potentials; these are rapid changes in the membrane potential enabling the transmission of e.g. neural signals.  The precise structural changes occurring in VGSCs are not completely understood, however.  Now, Ayumi Sumino and Takashi Sumikama from WPI-NanoLSI, Kanazawa University in collaboration with Katsumasa Irie from Wakayama Medical University and colleagues have succeeded in observing the structural dynamics of VGSC by means of high-speed atomic force microscopy (HS-AFM), a method capable of imaging the nanostructure and subsecond dynamics of biomolecules. 

VGSCs can be in three different states: resting, inactive and active.  In the latter state, Na+ ions can pass through the channel; in the resting and inactive states, which are structurally different, ions cannot pass.  The basic structure of a VGSC consists of two modules: voltage sensor domains and pore domains.  These domains form a square arrangement, with the ion pore at its center.  An important open question is whether the voltage sensor domains dissociate from the pore domains when the channel closes.

Sumino and colleagues performed experiments on three VGSCs.  One is the sodium channel of a particular bacterium (Arcobacter butzleri), the other two are mutants thereof.  These three VGSCs have different voltage dependencies, with activation voltages starting at -120 mV, -50 mV and 0 mV, so that at the experimental conditions (0 mV), the VGSCs are in different states. 

In order to provide insights into the structural dynamics of these three VGSCs, the researchers applied HS-AFM, a powerful technique for producing image sequences of biochemical compounds.  A single AFM image is generated by laterally moving a tip just above the sample’s surface; during this xy-scanning motion, the tip’s position in the direction perpendicular to the xy-plane (the z-coordinate) will follow the sample’s height profile.  The variation of the z-coordinate of the tip then produces a height map — the image of the sample.  The generation of such AFM images in rapid succession then produces a video recording of the sample. 

The HS-AFM results revealed that for the mutant VGSC in the resting state, the voltage sensor domains are indeed dissociated from the pore domains.  Furthermore, the researchers found that the dissociated voltage sensor domains of neighboring channels connect to form pairs — this is referred to as dimerization. 

The observation of the dissociation of voltage sensor domains, as well as the dimerization between pore channels, are findings that will lead to a better understanding of what causes pores to close in the resting state, and how the development of action potentials is regulated.  Quoting the scientists, dimerization offers “a potential explanation for the facilitation of positive cooperativity of channel activity in the rising phase of the action potential”.



High-speed atomic force microscopy

The principle behind atomic force microscopy (AFM) is to scan the surface of a sample with a very small tip.  During this horizontal (xy) scanning, the tip, which is attached to a small cantilever, follows the sample’s vertical (z) profile, which induces a force on the cantilever that can be measured.  The magnitude of the force at the xy position can be related to the z value.  The xyz data generated during a scan then result in a height map providing structural information about the investigated sample.  In high-speed-AFM (HS-AFM), the working principle is slightly more involved: the cantilever is made to oscillate near its resonance frequency.  When the tip is moved around a surface, the variations in the amplitude (or the frequency) of the cantilever’s oscillation — resulting from the tip’s interaction with the sample’s surface — are recorded, as these provide a measure for the local z value.

HS-AFM produces a sequence of AFM images recorded in rapid succession — a video, where the time interval between frames depends on the speed with which a single image can be generated (by xy-scanning the sample).  In recent years, researchers at WPI-NanoLSI of Kanazawa University have further developed HS-AFM so that it can be applied for studying biochemical molecules and biomolecular processes in real-time.  Ayumi Sumino and colleagues have now applied the method to study the structural dynamics of Na+ ion channels, revealing the dissociation of channel components and the formation of inter-channel dimers when the channel is in the resting (blocking) state.

About the World Premier International Research Center Initiative (WPI)

 The WPI program was launched in 2007 by Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) to foster globally visible research centers boasting the highest standards and outstanding research environments. Numbering more than a dozen and operating at institutions throughout the country, these centers are given a high degree of autonomy, allowing them to engage in innovative modes of management and research. The program is administered by the Japan Society for the Promotion of Science (JSPS).
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Nano Life Science Institute (WPI-NanoLSI), Kanazawa University

Understanding nanoscale mechanisms of life phenomena by exploring “uncharted nano-realms”

Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale.
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Published: 18 Jan 2024


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Content type: 

Author: Ayumi Sumino, Takashi Sumikama, Mikihiro Shibata, and Katsumasa Irie.
Title: Voltage sensors of a Na+ channel dissociate from the pore domain and form inter-channel dimers in the resting state,
DOI: 10.1038/s41467-023-43347-3