The intracellular Ca ²⁺ release channel TRPML1 regulates lower urinary tract smooth muscle contractility

Abstract

TRPML1 (transient receptor potential mucolipin 1) is a Ca²⁺-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca²⁺ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (<i>Mcoln1</i>^<i>-/-</i> ) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca²⁺-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca²⁺ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca²⁺ ("Ca²⁺ sparks") that activate plasmalemmal large-conductance Ca²⁺-activated K⁺ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in <i>Mcoln1</i>^<i>-/-</i> mice, which showed diminished spontaneous Ca²⁺ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca²⁺ spark-BK channel signaling in <i>Mcoln1</i>^<i>-/-</i> mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in <i>Mcoln1</i>^<i>-/-</i> mice. We conclude that TRPML1 is critically important for Ca²⁺ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.