COMMENTARY COMMENTARY

+ Igniting Ca2 sparks with TRPML1 Gerard P. Sergeanta,1, Mark A. Hollywooda, and Keith D. Thornburya

Storage and voiding of urine in mammals is accom- leading to hyperpolarization of membrane potential + plished by a reciprocal contractile relationship be- and SMC relaxation (8, 10). Ca2 sparks originate from + tween the bladder and the urethra. During the storage ryanodine-sensitive intracellular Ca2 release channels phase, the urethra remains contracted to prevent (known as ryanodine receptors [RyRs]) on the sarco- leakage of urine, while the bladder is relaxed to accom- plasmic reticulum (SR) membrane (11). RyRs are acti- + modate the increased volume of urine. Conversely, vated by micromolar levels of intracellular Ca2 via + + during voiding, the urethra relaxes, while the bladder Ca2 -induced Ca2 release (CICR). In cardiac myo- + contracts to generate an intravesical pressure which cytes, RyR activation is tightly coupled to Ca2 influx exceeds that in the urethra (1). The cellular mecha- via VDCC (11); however, this is not the case in smooth nisms that govern these functions are only partly under- muscle, where there is only “loose coupling” between stood, and there is a particular paucity of data on plasmalemmal VDCCs and RyRs (12). the cellular processes that underlie tonic contraction The mechanisms responsible for the localized ac- + of urethral smooth muscle (USM). This issue is addressed tivation of RyRs that initiate Ca2 sparks in detrusor in PNAS by Griffin et al. (2), who demonstrate a crit- and urethral myocytes have remained elusive. This is- ical role for transient receptor potential mucolipin 1 sue is addressed by Griffin et al. (2), who investi- + (TRPML1) channels in the regulation of detrusor and gate whether TRPML1 channels, a subfamily of Ca2 USM excitability. -permeable, nonselective cation channels located on Spontaneous phasic contractions of the detrusor the membrane of late endosomes and lysosomes + are directly correlated with bursts of action potentials, (LELs), are involved in the generation of Ca2 sparks,

mediated by activation of voltage-dependent CaV1.2 by inducing CICR via RyRs. Traditionally, intracellular + channels (VDCC) (3). Selective blockade of large con- release of Ca2 through TRPML1 channels is thought + + ductance Ca2 -activated K (BK) channels with iberio- to be involved in the regulation of intravesicular pH, toxin or paxilline induced depolarization and increased as well as the maturation, fusion, and trafficking of the amplitude and duration of spontaneous action po- endolysosomes (13); therefore, the idea that TRPML1 + tentials and phasic contractions (4). Furthermore, channels could trigger Ca2 sparks is not immediately transgenic mice that lack the BK channel α subunit dem- intuitive. However, two lines of evidence led Griffin onstrate a marked increase in urination frequency, corre- et al. to pursue this hypothesis. Firstly, Thakore et al. + sponding to an overactive bladder (OAB) phenotype (5). (14) reported that Ca2 release via TRPML1 channels + + This is thought to result from increased Ca2 influx via was pivotal for the generation Ca2 sparks in vascu- VDCC as a result of diminished negative feedback, lar smooth muscle, and, secondly, genetic ablation + − − which limits Ca2 entry. The BK channel blocker, peni- of TRPML1 in mice (Mcoln1 / ) induced a hyperdis- trem A, increased the amplitude and duration of action tended/hypertrophic bladder phenotype, indicative potentials in USM cells (USMC) (6, 7), indicating that BK of a role in detrusor contraction (15). Triggering of + + channels are also important regulators of USM excitability. Ca2 release from RyRs on the SR by Ca2 efflux via The primary activation mechanism for BK channels TRPML1 channels on LEL implies that these pro- + in detrusor myocytes is localized Ca2 release from teins form stable signaling complexes and therefore + intracellular stores, referred to as Ca2 sparks (8). that the LELs that form these complexes are immo- The resultant BK currents are termed spontaneous bile. This was confirmed using four-dimensional lattice transient outward currents (STOCs) and were first light-sheet live-cell microscopy of isolated detrusor reported in intestinal myocytes (9). In the detrusor, a myocytes loaded with a late endosome-specific fluo- + single Ca2 spark can activate up to 100 BK channels, rescent dye (LysoTracker Red).

aSmooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk A91 K584, Ireland Author contributions: G.P.S., M.A.H., and K.D.T. wrote the paper. The authors declare no competing interest. Published under the PNAS license. + See companion article, “The intracellular Ca2 release channel TRPML1 regulates lower urinary tract smooth muscle contractility,” 10.1073/pnas. 2016959117. 1To whom correspondence may be addressed. Email: [email protected]. First published December 1, 2020.

32836–32838 | PNAS | December 29, 2020 | vol. 117 | no. 52 www.pnas.org/cgi/doi/10.1073/pnas.2022896117 Downloaded by guest on October 1, 2021 + + Fig. 1. Ca2 efflux via TRPML1 channels in late endosomes and lysosomes creates a Ca2 microdomain in the vicinity of neighboring RyR2 + + channels on the SR that triggers Ca2 sparks via CICR. Consequently, the initial TRMPL1-mediated Ca2 release event is amplified by RyR2s to + generate a Ca2 spark that activates BK channels on the plasma membrane.

− − Three RyR subtypes have been identified (RyR1 to RyR3), and, activity observed in Mcoln1 / mice would be expected to in- while transcripts for all three subtypes have been detected in the crease detrusor contractility, as is the case in Kcnma1- and RyR2- detrusor, it is the RyR2 isoform that is functionally important in the deficient mice (5, 8). Using isometric tension recordings, Griffin + generation of Ca2 sparks (10). Using superresolution microscopy, et al. (2) show that spontaneous and carbachol-induced contrac- − − Griffin et al. (2) demonstrate that RyR2 channels on the SR coloc- tions of bladder strips from Mcoln1 / mice were greatly enhanced, alize with TRPML1 channels and Lamp-1 (lysosomal-associated demonstrating the importance of the TRPML1:RyR2:BK signaling membrane protein 1) on LEL. These proteins are present as de- complex in regulating detrusor contraction. − − fined clusters, with a substantial fraction of Lamp-1–localized The hyperdistended bladders in Mcoln1 / mice are reminis- TRPML1 channels located within 40 nm of RyR2 clusters. This is cent of those observed in murine models of bladder outlet ob- consistent with the idea that TRPML1 channels, located on sta- struction (16) and in transgenic mice lacking nitric oxide synthase tionary LELs, form stable signaling complexes with RyR2. (17). This phenotype is consistent with an impairment in the ability The functional relevance of this signaling complex was assayed of the urethra to relax properly and suggests that TRPML1 chan- using several experimental approaches (2). Firstly, imaging with nels could also regulate USM contraction. Since USM excitability + the Ca2 -sensitive fluorophore (Fluo4 AM) revealed that, in con- is also regulated by BK channel activity (6, 7), Griffin et al. (2) − − + trast to detrusor myocytes from wild-type (WT) mice, Mcoln1 / examined whether Ca2 sparks and BK-mediated STOCs in USMC + − − detrusor myocytes fired fewer Ca2 sparks (10% of Mcoln1 / were also reduced by genetic ablation of TRPML1 channels. They + cells, compared to 90% of controls). This did not appear to be found that USMC isolated from WT mice generated Ca2 sparks due to direct dysfunction of RyRs, as caffeine responses remained that were inhibited by ryanodine. However, this activity was − − intact and RyR2 expression levels were the same as WT controls. greatly reduced in USMC isolated Mcoln1 / mice. Similarly, the + The next step was to examine whether loss of Ca2 sparks in frequency and amplitude of STOCs in USMC isolated from − − TRPML1-deficient mice translated to reduced STOC activity. Mcoln1 / mice was also greatly reduced in comparison to WT Detrusor myocytes from WT mice exhibited robust STOC activity, controls. These observations suggest that the TRPML1:RyR:BK − − whereas STOCs in cells isolated from Mcoln1 / mice were signaling complex also modulates USM contraction. This was also greatly reduced in frequency. Furthermore, application of the supported by the finding that phenylephrine-induced contrac- − − TRPML1 agonist, ML-SA1, increased STOC frequency significantly tions of USM strips, taken from Mcoln1 / mice, were larger than − − in WT mice, but had no effect in Mcoln1 / mice. These effects are those in WT controls. Therefore, it appears that TRPML1 channels not attributable to BK channel dysfunction or expression, as there regulate contractility of both bladder and USM and that the hyper- − − is no difference in BK current density (pA/pF) or Kcnma1 and distended bladder phenotype observed in Mcoln1 / mice is Kcnmb1 expression (genes encoding the BKα pore-forming sub- likely to result from altered contraction of both tissues. Cystometry − − unit and BKβ1 regulatory subunit, respectively) between WT and experiments on Mcoln1 / mice showed that they had a signifi- − − + Mcoln1 / detrusor. These data suggest that opening of Ca2 cant increase in voiding frequency in comparison with WT litter- -permeable TRPML1 channel clusters on the membrane of LEL mates, consistent with symptoms of OAB. OAB symptoms in + activates nearby RyR2 on the SR via CICR, triggering Ca2 sparks these mice are likely to result from a combination of direct en- + and BK-mediated STOCs. The reduced Ca2 spark and BK STOC hancement of bladder contractility and an enhancement that is

Sergeant et al. PNAS | December 29, 2020 | vol. 117 | no. 52 | 32837 Downloaded by guest on October 1, 2021 + secondary to augmented USM contraction resulting in bladder plasmalemmal Ca2 -dependent channels, such as TMEM16A 2+ hypertrophy. channels, or intracellular Ca release channels, such as IP3Rs. The study by Griffin et al. (2) identifies TRPML1 channels on This study (2) also raises the possibility that TRPML1 channels + LEL as an essential component of the Ca2 spark/BK signaling could be targeted for treatment of lower urinary tract (LUT) disorders pathway in bladder and urethral myocytes. Opening of TRPML1 such as OAB or benign prostatic hyperplasia (BPH). Is TRPML1 ex- + channels is proposed to create a Ca2 microdomain in the vicinity pression, or function, altered in patients with such disorders? Would + of neighboring RyR2 channels on the SR that triggers Ca2 sparks a TRPML1 activator relax USM and relieve urinary retention in pa- + via CICR. Consequently, the initial TRPML1-mediated Ca2 re- tients with BPH or inhibit contractions of the bladder that lead to + lease event is amplified by RyR2s to generate a Ca2 spark that symptoms of urge in patients with OAB? Alternatively, could a activates BK channels (Fig. 1). This work raises many important TRPML1 inhibitor be used to increase urethral tone and closure pres- questions about the overall physiological regulation of this path- sure in patients with stress urinary incontinence? Ultimately, the ther- way and its relevance to other cellular processes. For example, it apeutic use of TRPML1 modulators for LUT disorders would rely on would be useful to know how TRPML1 activity is regulated and the ability to selectively target TRPML1 channels in a tissue-specific + how lysosomes refill with Ca2 , as this could be a key determinant manner. The findings presented by Griffin et al. suggest that TRPML1 + of Ca2 spark frequency. It would also be of interest to establish channels play an important role in LUT smooth muscle contraction + whether Ca2 release from TRPML1 channels can affect other and represent a potential therapeutic target for LUT disorders.

1 A. F. Brading, The physiology of the mammalian urinary outflow tract. Exp. Physiol. 84, 215–221 (1999). + 2 C. S. Griffin et al., The intracellular Ca2 release channel TRPML1 regulates lower urinary tract smooth muscle contractility. Proc. Natl. Acad. Sci. U.S.A. 117, 30775–30786 (2020). 3 H. Hashitani, A. F. Brading, H. Suzuki, Correlation between spontaneous electrical, calcium and mechanical activity in detrusor smooth muscle of the guinea-pig bladder. Br. J. Pharmacol. 141, 183–193 (2004). 4 G. V. Petkov, Central role of the BK channel in urinary bladder smooth muscle physiology and pathophysiology. Am. J. Physiol. Regul. Integr. Comp. Physiol. 307, R571–R584 (2014). 5 A. L. Meredith, K. S. Thorneloe, M. E. Werner, M. T. Nelson, R. W. Aldrich, Overactive bladder and incontinence in the absence of the BK large conductanceCa2+- activated K+ channel. J. Biol. Chem. 279, 36746–36752 (2004). + 6 M. A. Hollywood, K. D. McCloskey, N. G. McHale, K. D. Thornbury, Characterization of outward K currents in isolated smooth muscle cells from sheep urethra. Am. J. Physiol. Cell Physiol. 279, C420–C428 (2000). 7 B. Kyle et al., Contribution of Kv2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra. Am. J. Physiol. Cell Physiol. 301, C1186–C1200 (2011). + 8 Y. Ohi et al., Local Ca2 transients and distribution of BK channels and ryanodine receptors in smooth muscle cells of guinea-pig vas deferens and urinary bladder. J. Physiol. 534, 313–326 (2001). 9 C. D. Benham, T. B. Bolton, Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit. J. Physiol. 381, 385–406 (1986). 10 S. Hotta et al., Ryanodine receptor type 2 deficiency changes excitation-contraction coupling and membrane potential in urinary bladder smooth muscle. J. Physiol. 582, 489–506 (2007). 11 J. H. Jaggar, V. A. Porter, W. J. Lederer, M. T. Nelson, Calcium sparks in smooth muscle. Am. J. Physiol. Cell Physiol. 278, C235–C256 (2000). 12 M. L. Collier, G. Ji, Y. Wang, M. I. Kotlikoff, Calcium-induced calcium release in smooth muscle: Loose coupling between the action potential and calcium release. J. Gen. Physiol. 115, 653–662 (2000). 13 K. Venkatachalam, C. O. Wong, M. X. Zhu, The role of TRPMLs in endolysosomal trafficking and function. Cell Calcium 58,48–56 (2015). + 14 P. Thakore et al., TRPML1 channels initiate Ca2 sparks in vascular smooth muscle cells. Sci. Signal. 13, eaba1015 (2020). 15 B. Venugopal et al., Neurologic, gastric, and opthalmologic pathologies in a murine model of mucolipidosis type IV. Am. J. Hum. Genet. 81, 1070–1083 (2007). 16 A. Schröder, B. Uvelius, D. Newgreen, K. E. Andersson, Bladder overactivity in mice after 1 week of outlet obstruction. Mainly afferent dysfunction? J. Urol. 170, 1017–1021 (2003). 17 A. L. Burnett et al., Urinary bladder-urethral sphincter dysfunction in mice with targeted disruption of neuronal nitric oxide synthase models idiopathic voiding disorders in humans. Nat. Med. 3, 571–574 (1997).

32838 | www.pnas.org/cgi/doi/10.1073/pnas.2022896117 Sergeant et al. Downloaded by guest on October 1, 2021