Journal Club: Demonstration of Human TMC1 and TMC2 as stretch-activated ion channels, enabled by knockdown of UROD in HEK293T cells.

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Today's journal article

Fu S, Pan X, Lu M, Dong J, Yan Z. Human TMC1 and TMC2 are mechanically gated ion channels. 

Why I picked this article

The abstract says it all for this article... But this research shines light on the functional identity of proteins called TMC1 and TMC2, which are thought to be critical for our sense of hearing. 

Our inner ear can detect sounds because we have sensory cells for hearing called hair cells. Hair cells have "hair" or "stereocilia" as a specialised compartment within the hair cell. Bending of stereocilia causes ions to flow into the hair cells, causing them to, in turn, release chemicals at synapses and activate the auditory nerve. 

It has long been thought that the ions flow into hair cells via proteins called transmembrane channel-like protein 1 and 2, or TMC1 and TMC2, found on the stereocilia. However, this has not been fully "demonstrated" to be the case because of technical difficulty in testing this in experiments. Typically, to show the functional identity of a protein, researchers use genetic modification to produce a lot of the target protein in cultured cells and conduct experiments. For TMC1 and TMC2, this strategy has not worked because when TMC1/TMC2 are produced in cultured cells, they tend not to move to the correct location within the cell. 

This research overcame such a challenge by a series of highly technical molecular biology techniques, ultimately finding a way to move TMC1 and TMC2 to the cell membrane in cultured cells, to demonstrate the function of TMC1 and TMC2. 

Some of the research findings

  • OSCA protein has some similarities in protein structure to TMC1 and 2. The Chimaera gene was produced so that some parts of TMC1 and 2 were replaced with similar parts of OSCA1.1. Swapping was done for the region of proteins called "TM3-TM7" to compare which "TM" made a difference. Human cultured cell line, HEK293 cells, were used. 
  • Initial testing with OSCA1.1 chimaera protein showed that swapping the "TM3" region of TMC1/2 with OSCA1.1 enabled the chimaera protein to locate itself to the cell membrane. 
  • Researchers then focused on 36 amino acids within "TM3" region of TMC1/2, and tested modification one by one by point mutation. They identified mutated versions of TMC1/2, TMC1-H353A and TMC2-H406A, which could be localised to the cell membrane and function as ion channels. 

Part of Figure 1E. Green - inside the cell, Red - chimera protein. Fu et al. (2025)

  • Researchers next used original (or wild-type) human TMC2 and produced a modified HEK293 cell line which produces TMC2. TMC2-Cas9-KRAB HEK293T cell line, screening experiments were done by applying the "genome-wide CRISPRi screening" methodology. In brief, this allows massive screening out of many genes (templates for many proteins) to identify which gene has the most impact. The screening identified three candidate proteins, ARL1, RGP1, and UROD, as proteins which affect the locations of TMC1/2. 
  • Finally, researchers found that if you reduce the amount of UROD, the original form of TMC1/2 can be found in the cell membrane. Using this special model, they show that TMC1 and 2 function as stretch-activated ion channels, where stretching of the cell membrane can open TMC1 and 2 as channels, and allow movement of ions through them. 

Haruna's takeaway

This was a massive paper with a huge determination to get to the bottom of TMC1/TMC2 molecular biology. All point mutations, transfections and screenings are highly technical molecular biology, and a combination of all the effort enabled the end results of not only demonstrating TMC1 and TMC2 function as the ion channels, but also identifying some mechanism related to how TMC1 and 2 are stopped from being located at the cell membrane by UROD. 

What I have learned from this publication is the screening strategy, going from TM3-7 down to TM3 first with the chimaera protein, point mutations to narrow down to amino acids, in parallel to using genome-wide screening to identify what's stopping TMC1/2 from working in some cultured cells. It is a very nice demonstration of how a massive effort with a correct screening process can narrow down your hypothesis and ultimately land on some answer. It raises a lot of interesting questions about the complexity of how proteins are moved around within the cell more broadly. 


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This is Haruna's 20/100 of the 100-day challenge to post a science blog article every day! I love inner ear biology & cochlear physiology.