Galvan, C. | bioRxiv Science

bioRxiv2026

Deep mutational scan of the pore of the cold-sensing TRPM8 channel

Members of the Transient Receptor Potential (TRP) family of ion channels have a nearly ubiquitous role in human physiology, tuning cell signaling to remarkably diverse physical and chemical stimuli. Although there is extensive structural data on TRP channels, a systematic and unbiased interrogation of structure-function relations in these proteins is required to fully elucidate their mechanisms of function. By focusing on a critical pore region of the TRPM8 channel, which is the main detector of cold and cooling agents in sensory neurons, we show how deep mutational scanning can be used in combination with the available structural data to understand how TRP channels respond to stimuli. We define a novel mechanism whereby the extracellular pore loop, which has only been resolved in structures representing desensitized states of the channel, plays an essential role in the response of TRPM8 to menthol or cold by coordinating the movement of the S6 helices that line and gate the pore, and the ion-selectivity filter that binds permeant cations. Moreover, our screen reveals sequence determinants along the S6 helices that explain how their architecture sustains gating and, together, provide strong support for a structural mechanism of TRPM8 pore opening in response to menthol and cold.

biophysics↗

bioRxiv2023

Defining metabolic flexibility in hair follicle stem cell induced squamous cell carcinoma

Among the numerous changes associated with the transformation to cancer, cellular metabolism is one of the first discovered and most prominent[1, 2]. However, despite the knowledge that nearly every cancer is associated with the strong upregulation of various metabolic pathways, there has yet to be much clinical progress on the treatment of cancer by targeting a single metabolic enzyme directly[3-6]. We previously showed that inhibition of glycolysis through lactate dehydrogenase (LDHA) deletion in cancer cells of origin had no effect on the initiation or progression of cutaneous squamous cell carcinoma[7], suggesting that these cancers are metabolically flexible enough to produce the necessary metabolites required for sustained growth in the absence of glycolysis. Here we focused on glutaminolysis, another metabolic pathway frequently implicated as important for tumorigenesis in correlative studies. We genetically blocked glutaminolysis through glutaminase (GLS) deletion in cancer cells of origin, and found that this had little effect on tumorigenesis, similar to what we previously showed for blocking glycolysis. Tumors with genetic deletion of glutaminolysis instead upregulated lactate consumption and utilization for the TCA cycle, providing further evidence of metabolic flexibility. We also found that the metabolic flexibility observed upon inhibition of glycolysis or glutaminolysis is due to post-transcriptional changes in the levels of plasma membrane lactate and glutamine transporters. To define the limits of metabolic flexibility in cancer initiating hair follicle stem cells, we genetically blocked both glycolysis and glutaminolysis simultaneously and found that frank carcinoma was not compatible with abrogation of both of these carbon utilization pathways. These data point towards metabolic flexibility mediated by regulation of nutrient consumption, and suggest that treatment of cancer through metabolic manipulation will require multiple interventions on distinct pathways.

cancer biology↗