TRPML (transient receptor potential mucolipin) are a group of calcium (Ca²⁺)-permeable, nonselective cation channels primarily found in the membranes of late endosomes and lysosomes. The release of Ca²⁺ through TRPML is believed to be crucial for maintaining the acidic pH inside vesicles, as well as for the maturation, fusion, and trafficking of lysosomes (Venkatachalam et al., 2015). Recently, TRPML channels subtype 1 (TRPML1) have been shown to regulate vascular, bladder and urethral smooth muscle contractility and excitability (Thakore et al., 2019; Griffin et al., 2020). TRPML1 activity is thought to exert its regulatory effect on these tissues, by initiating Ca²⁺ sparks from ryanodine receptors (RyR), via a Ca²⁺-induced Ca²⁺ release (CICR) mechanism. TRPML1-mediated Ca²⁺ spark activity leads to BK channel activation, membrane potential hyperpolarization, and smooth muscle cell (SMC) relaxation. In the proximal colon, Ca²⁺ signaling is pivotal for the regulation of smooth muscle contractility, however the role of TRPML channels in mediating these Ca²⁺ signaling pathways and/or muscle contractility has yet to be explored. Therefore, our aim was to investigate the role of TRPML channels in regulating proximal colon contractility.

Using isometric tension of murine proximal colon circular muscle strips, we found that the proximal colon displayed robust spontaneous contractile activity, similar to previous studies (Drumm et al,. 2019; Koh et al., 2022). Application of a selective TRPML agonist, ML-SA1, increased both the frequency (contractions min^-1) and tension (Area; mN.s) of proximal colon spontaneous contractions in a concentration dependent manner (100nM to 10µM). For example, 10µM of MLSA1 increased contraction frequency by 475% and contraction tension by 151% compared to control activity (n=2). Interestingly, application of a selective TRPML antagonist, ML-SI3, mimicked the effect of the agonist, ML-SA1, by increasing both contraction frequency and contraction tension (n=2). Tonic release of Nitric oxide (NO) from inhibitory motor neurons has been shown to suppress proximal colon contractility. The influence of tonically released NO on contractile activity can be removed by inhibiting nerves with tetrodotoxin (TTX; Drumm et al., 2019). We found that application of TTX (300 nM) increased spontaneous contractile activity of the proximal colon. However, when we applied the TRPML agonist, ML-SA1, in the presence of TTX, spontaneous contractile activity decreased in a concentration dependent manner (100nM to 10µM). For example, application of 10µM of MLSA1 decreased contraction tension by 67% in comparison to control (n=2). The TRPML antagonist, ML-SI3, also reduced contraction tension in the presence of TTX, however to a lesser extent (32%; n=2).

Taken together, our data suggest that TRPML channels regulate proximal colon activity, however it appears that a complex relationship exists between TRPML channels, NO-releasing inhibitory nerves and the excitability of the smooth muscle tissue. In the proximal colon, other cell types such as interstitial cells of cajal (ICC) and platelet derived growth factor receptor alpha (PDGFRα) cells also influence the excitability of the muscle and may express TRPML channels. Therefore, future studies should elucidate the precise cell type (s) and signaling pathway (s) that TRPML is exerting its effect on in the proximal colon.