From muscle contraction to nerve conduction, a wide range of physiological functions rely on ion channels, transmembrane proteins that shuttle charged species into and out of the cell. While these channels possess a well defined pore space through which ions travel, prolonged exposure to heat or certain ligands causes the pore to enlarge. Ion channels from the transient receptor potential (TRP) and P2X families exhibit this pore dilation phenomenon, resulting in an increased flow of ions, loss of directionality in transport, and access by larger charged molecules.
Although intrigued, scientists have struggled to understand pore dilation from a structural standpoint and to harness it for therapeutic applications. Now, researchers at Weill Cornell Medicine have captured advanced microscopy images that reveal that the TRPV3 ion channel undergoes a striking conformational change that corresponds to its pore-dilated state (1). Their work yields new insights into the structural basis of pore dilation and paves the way for strategies for controlling this property to enhance drug delivery or treat TRPV3-linked diseases.
The team first overexpressed the TRPV3 protein in cells, extracted it, and reconstituted it in an artificial lipid-based system that mimics the cell membrane. They then imaged the membrane-embedded ion channel with an atomic force microscope, where a thin tip scans across the surface of the protein and reads its topographical features at the single-molecule level like a needle in a record player. Specifically, the researchers used high-speed atomic force microscopy to image the channel with a temporal resolution of approximately one second, producing movie-like sequences that depict its dynamic behavior under physiological conditions. “You watch molecules actually at work,” said Simon Scheuring, a biophysicist at Weill Cornell Medicine.
While analyzing the microscopy data, Shifra Lansky, a postdoctoral researcher in Scheuring’s lab, noticed that a small fraction of the TRPV3 channels existed as five-subunit, pentameric structures rather than in the typical four-subunit, tetrameric conformation. The high-speed sequences showed that TRPV3 rearranges from tetramer to pentamer and back again by gaining or losing a monomer subunit. “That was the ultimate proof that these are indeed different states of the same channel, of the same molecule, and that they are interconvertible,” Scheuring said.
The researchers hypothesized that the pentamer represents the pore-dilated form of TRPV3. The transition between the tetrameric and pentameric states occurs on the timescale of seconds to minutes, consistent with the unusually slow kinetics of pore dilation. Furthermore, previous studies demonstrated that the higher the expression of ion channels in the cell membrane, the more likely the channels are to undergo pore dilation (2). Based on their proposed mechanism, the same would hold true for TRPV3, as a higher concentration of the tetramer would increase the probability that a monomer bumps into it to form a pentamer.
To gather more evidence, the team exposed TRPV3 to a ligand known to induce pore dilation and saw that it increased the percentage of channels in the pentameric state. Finally, they flash froze a sample of TRPV3 and imaged it by cryogenic electron microscopy to generate three-dimensional structural maps of the protein in its pentameric and tetrameric forms. They found that the pentamer pore is approximately 2.5-fold larger in diameter than the tetramer pore in its open state. “When you see the pentamer with this huge pore, it just jumps to your eye,” Scheuring said. “An image speaks more than a thousand words.”
When you see the pentamer with this huge pore, it just jumps to your eye. ...An image speaks more than a thousand words.
- Simon Scheuring, Weill Cornell Medicine
The researchers are now working on computationally solving the structure of the pentameric TRPV3 channel in higher resolution. In this way, “we could understand what the residues are that are crucial for formation of the pentamer versus the tetramer,” Lansky said. With this information, scientists may be able to design compounds that bind to specific residues to inhibit TRPV3 pore dilation when it is implicated in disease.
For example, the rare skin disease Olmsted syndrome is characterized by a TRPV3 mutation at the interface between the protein’s subunits (3). Scheuring and Lansky hypothesize that this mutation may disrupt the interaction between the subunits, destabilizing the tetramer and promoting conversion to the pentamer. As TRPV3 helps to regulate several cellular processes in the skin, this pore dilation could lead to dysfunctional ion transport in Olmsted syndrome, providing a promising application for inhibitors.
A high-resolution structure could also guide the development of compounds that activate the formation of the TRPV3 pentamer to facilitate drug delivery. Such a molecule could be administered alongside a hydrophilic drug that cannot penetrate the cell membrane, enabling the drug to pass through the enlarged pore of the ion channel. To avoid inducing pore dilation in the absence of the drug, the activator could be designed with a defined lifespan, allowing the pentamer to return to its native conformation once the compound has degraded. “The fact that we see that this pentamer can reversibly turn into a tetramer is in our favor,” Lansky said.
References
- Lansky, S. et al. A pentameric TRPV3 channel with a dilated pore. Nature 621, 206-214 (2023).
- Chung, M.K., Güler, A.D., & Caterina, M.J. Biphasic currents evoked by chemical or thermal activation of the heat-gated ion channel, TRPV3. J Biol Chem 280, 15928-15941 (2005).
- Ni, C. et al. A novel mutation in TRPV3 gene causes atypical familial Olmsted syndrome. Sci Rep 6, 21815 (2016).
