Abstract Non‐selective cation channels in urinary bladder smooth muscle (UBSM) are thought to mediate increases in cellular excitability and contractility. For transient receptor potential melastatin type‐4 (TRPM4) channels, the evidence primarily relies on the inhibitor 9‐phenanthrol, which exhibits pharmacological limitations. Recently, 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA) has been discovered as a novel TRPM4 channel blocker. We examined how, in comparison to 9‐phenanthrol, CBA affects the excitability of freshly isolated guinea pig UBSM cells and the contractility of UBSM strips. Additionally, non‐selective TRPM4 channel inhibitor flufenamic acid (FFA) and potentiator BTP2 (also known as YM‐58483) were studied in UBSM cells. Unlike robust inhibition for 9‐phenanthrol already known, CBA (up to 100 μM) displayed either no or a very weak reduction (<20%) in spontaneous phasic, 20 mM KCl‐induced, and electrical field stimulated contractions. For 300 μM CBA, reductions were higher except for an increase in the frequency of KCl‐induced contractions. In UBSM cells, examined under amphotericin B‐perforated patch‐clamp, CBA (30 μM) did not affect the membrane potential (I = 0) or voltage step‐induced whole‐cell cation currents, sensitive to 9‐phenanthrol. The currents were not inhibited by FFA (100 μM), increased by BTP2 (10 μM), nor enhanced under a strongly depolarizing holding voltage of −16 or + 6 mV (vs. −74 mV). None of the three compounds affected the cell capacitance, unlike 9‐phenanthrol. In summary, the novel inhibitor CBA and nonselective FFA did not mimic the inhibitory properties of 9‐phenanthrol on UBSM function. These results suggest that TRPM4 channels, although expressed in UBSM, play a distinct role rather than direct regulation of excitability and contractility.

During development, maturation, or aging, the expression and function of urinary bladder smooth muscle (UBSM) ion channels can change, thus affecting micturition. Increasing evidence supports a novel role of transient receptor potential melastatin‐4 (TRPM4) channels in UBSM physiology. However, it remains unknown whether the functional expression of these key regulatory channels fluctuates in UBSM over different life stages. Here, we examined TRPM4 channel protein expression (Western blot) and the effects of TRPM4 channel inhibitors, 9‐phenanthrol and glibenclamide, on phasic contractions of UBSM isolated strips obtained from juvenile (UBSM‐J, 5–9 weeks old) and adult (UBSM‐A, 6–18 months old) male guinea pigs. Compared to UBSM‐J, UBSM‐A displayed a 50–70% reduction in total TRPM4 protein expression, while the surface‐to‐intracellular expression ratio (channel trafficking) remained the same in both age groups. Consistent with the reduced total TRPM4 protein expression in UBSM‐A, 9‐phenanthrol showed lower potencies and/or maximum efficacies in UBSM‐A than UBSM‐J for inhibiting amplitude and muscle force of spontaneous and 20 mM KCl‐induced phasic contractions. Compared to 9‐phenanthrol, glibenclamide also attenuated both spontaneous and KCl‐induced contractions, but with less pronounced differential effects in UBSM‐A and UBSM‐J. In both age groups, regardless of the overall reduced total TRPM4 protein expression in UBSM‐A, cell surface TRPM4 protein expression (~80%) predominated over its intracellular fraction (~20%), revealing preserved channel trafficking mechanisms toward the cell membrane. Collectively, this study reports novel findings illuminating a fundamental physiological role for TRPM4 channels in UBSM function that fluctuates with age.

Introduction Voltage-gated K+ (KV) channels, specifically type 7 (KV7), are emerging key regulators of urinary bladder smooth muscle (UBSM) excitability and contractility. The KV7 channel family consists of 5 pore-forming α-subunits (KV7.1–KV7.5) capable of assembling as homotetrameric (e.g. KV7.4) or heterotetrameric (e.g. KV7.4/KV7.5) channels; thus, further increasing species/tissue expression diversity and regulatory potential. However, much remains unknown about KV7 channel properties in UBSM and the translational value of the common experimental animal model - the rat. Hypothesis KV7 channels, especially homomeric or heteromeric KV7.4 and KV7.5 subtypes, regulate rat UBSM excitability and contractility. Methods UBSM tissue without mucosa was obtained from adult male Sprague Dawley rats. UBSM homogenates were used for Western blots to detect KV7 channel subtypes with or without surface biotinylation. UBSM isometric tension recordings were carried out in the presence of tetrodotoxin for spontaneous, 20 mM K+-induced and 1 µM carbachol-induced contractions or in its absence for the electrical field stimulated (EFS) contractions. For electrophysiological amphotericin-B perforated patch-clamp studies, single UBSM cells were prepared using enzymatic dissociation. UBSM cell membrane potential was recorded in current-clamp (I=0) mode at room temperature. The following KV7 channel modulators were tested: XE991 (KV7.1-KV7.5 subtype blocker), retigabine (KV7.2-KV7.5 activator), and ICA-069673 (selective activator for KV7.2/KV7.3, which at higher concentrations preferentially enhances KV7.4 and KV7.4/KV7.5 but not KV7.5 channels). Results Western blot experiments detected KV7.4 and KV7.5 channels in UBSM under both non- and surface-biotinylation assay conditions. The latter condition revealed that for both KV7.4 and KV7.5 subtypes surface localization predominated (>75 %). In isometric tension experiments, retigabine attenuated contractility in a concentration-dependent manner for all four contraction protocols (EC50 values: 2.6-58.7 µM for the amplitude and force, with complete inhibition of contractility at 100 µM, n=3-10). XE991 (10 µM) depolarized the membrane potential of isolated UBSM cells (Δ10.5±3.6 mV, n=5, p<0.05). In contrast, retigabine (10 µM, Δ-6.5±1.8 mV, n=4, p<0.04) and ICA-069673 (10 µM, Δ-11.5±2.4 mV, n=4, p<0.02), when applied separately, induced UBSM cell hyperpolarization. In the presence of XE991, ICA-069673 (10 µM) did not significantly change the membrane potential of isolated UBSM cells (Δ0.4±1.2 mV, n=4, p>0.05). Conclusions Our data show that both KV7.4 and KV7.5 channel subtypes are expressed in rat UBSM and display preferential cell membrane localization. Pharmacological modulation of KV7 channels can control UBSM cell excitability and tissue contractility. The hyperpolarizing effect of ICA-069673, which is blocked by XE991, revealed a critical role of KV7.4 homomeric and/or KV7.4/KV7.5 heteromeric channels in UBSM function.

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 °C for 30-45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

Novel technologies facilitate breakthroughs in scientific discovery with concomitant advances in therapy. In the case of nanotechnology, a major focus has been on optimizing its use for targeted drug delivery, imaging, diagnosis, or a combination of therapeutics and diagnosis (“theranosis”). However, the application of nanotechnology in the research and treatment of benign urological pathologies remains underexplored. At our institution (“Einstein”), the research laboratory of Dr. Joel Friedman has developed a nanoparticledelivery system (the “Einstein” nanoparticle). We and others have applied this system to multiple research fields, including benign urology, as documented by >20 publications, several extramurally funded research projects, and licensing to a commercial entity. This nanoparticle-delivery system has intrinsic potential for modulation of its physicochemical properties, allowing use in a vast array of basic research and clinical conditions. However, the availability of these nanoparticle-delivery systems to the general urologic research community is currently limited by the absence of specific resource allocations for design and synthesis. This proposal addresses these limitations by establishing a P20 Resource Development Center with two primary goals: 1) to educate and promote the use of nanotechnology within the urologic basic and clinical research community, and 2) to create a resource development (research project) component in which the “Einstein” nanoparticle will be available for collaborative projects focused on benign urologic diseases. The proposed Resource Center will design and synthesize nanoparticles tailored to each research project until commercial entities assume this role. The Center will be highly synergistic, with investigators learning how nanotechnology can be applied to their specific field of research. Investigators will have access to resources and training, so they can apply the “Einstein” nanotechnology in their project. Investigators will require design and synthesis of novel nanoparticle formulations tailored to their specific research projects. This process would therefore lead to the development and expansion of novel nanoparticle formulations for a variety of benign urologic conditions. We anticipate that commercial entities will be positioned to synthesize nanoparticles within 2-4 years, eventually replacing the need for this P20 Resource Center. CAIRIBU P20 Exploratory Centers for Interdisciplinary Research in Benign Urology Duke University, Department of Mechanical Engineering and Materials Science 2019-2021; PI, Pei Zhong, PhD DUKE UNIVERSITY P20 EXPLORATORY CENTER FOR INTERDISCIPLINARY RESEARCH IN BENIGN UROLOGY Laser lithotripsy (LL) is the treatment of choice for urinary stone disease (USD), which is the second most costly urologic condition in the US with a healthcare cost over $2 billion annually. LL is typically performed using Holmium (Ho):YAG laser operating at wavelength () of 2.1 m with a pulse repetition frequency (F) < 10 Hz. In recent years, new Ho:YAG lasers and technologies, such as the Lumenis H120 with MOSES techno logy, have enabled LL at high power (120 W)/high frequency (80 Hz), while offering new treatment modes, such as dusting, popcorning and pop-dusting. In 2020, Olympus launched the Soltive SuperPulsed Thulium Fiber Laser (TFL), operating at  = 1.94 m with F > 200 Hz, further expanding the armaments for USD management. Despite the rapid technology advances and growing clinical enthusiasm about the new lasers, the fundamental knowledge of LL has not changed commensurately in the past two decades. The Duke University P20 Exploratory Center for Interdisciplinary Research in Benign Urology has created a comprehensive program to investigate the mechanism of stone damage in LL through a combination of experimentation and numerical modeling. This is an important endeavor because better understanding of the mechanism of stone destruction is the first step in developing improved and even less invasive surgical technologies for managing patients with USD. Most importantly, we have discovered that cavitation, i.e., the formation of an elongated vapor bubble at the laser fiber tip, plays a significant and, in some cases, even dominant role in stone damage. This finding is in distinct contradiction to the prevailing theory that stone damage in LL is predominantly produced by photothermal ablation. This paradigm-changing observation opens up opportunities to improve LL treatment strategy and patient outcome based on optimization of bubble dynamics, instead of maximizing laser energy delivery to the stone. The overarching goal of our P20 program is to promote multidisciplinary collaborations from basic to translational and clinical research applied to improve the efficiency and safety of LL treatment for USD. CAIRIBU P20 Exploratory Centers for Interdisciplinary Research in Benign Urology University of Tennessee Health Science Center 2019-2021 PI, Georgi Petkov, PhD NOVEL APPROACH IN UROLOGICAL RESEARCH REVEALS DIFFERENTIAL ION CHANNEL SUBCELLULAR LOCALIZATION IN HUMAN URINARY BLADDER SMOOTH MUSCLE M. Dennis Leo1, John Malysz1, Eric S. Rovner1,2, Robert Wake1,3, Wenkuan Xin1, Georgi V. Petkov1,3,4 1Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN; 2Department of Urology, Medical University of South Carolina, Charleston, SC; 3Department of Urology; and 4Department of Pharmacology, College of Medicine, University of Tennessee Health Science Center INTRODUCTION AND BACKGROUND: Contraction and relaxation of detrusor smooth muscle (DSM) control micturition. DSM cell excitability and contractility depend on synchronized activity of multiple ion channel types. Our group, in collaboration with urologists, has the unique advantage to study the expression, function, and regulation of human DSM ion channels. Here, we have focused on two key DSM channel families: the TRPM (activation causes membrane depolarization and contraction) and voltage-gated Kv7 (activation causes hyperpolarization and relaxation). To exert their regulatory role, these channels would be expected to be localized to the DSM plasma membrane but so far investigations confirming this are lacking. To test this hypothesis, we selected the three most important family member representatives, the TRPM4, Kv7.4 and Kv7.5 channels in order to investigate their cellular localization. METHODS: We have employed a novel technique called ‘surface biotinylation’ in conjunction with immunocytochemistry to examine the overall plasma membrane versus intracellular localization of these three ion channels. Human DSM tissue strips were incubated with non-cell permeable biotin tagged reagents that specifically bind to cysteine and lysine protein residues. Biotinylated surface proteins were then separated using avidin beads, eluted and Western blotting performed to determine the overall surface (plasma membrane) to intracellular localization of these channel proteins. Immuocytochemistry analyses for Kv7.4 and Kv7.5 channels were also performed on freshly isolated human DSM cells. RESULTS: Surface biotinylation revealed that >85% of total TRPM4 protein was localized to the surface of human DSM cells, with only ~15% appearing in the intracellular fraction. Similarly, >82% of total KV7.4 and ~66% of total Kv7.5 proteins were also localized on the surface of DSM cells. Interestingly, the Kv7.5 distribution surface/intracellular (~66%/34%) ratio was the lowest among the three channels studied. Immunocytochemistry data analyses revealed that Kv7.4 channels were predominantly surface localized while Kv7.5 displayed a uniform distribution. CONCLUSION: By employing surface biotinylation, a novel approach in urological research, along with immunocytochemistry, we revealed differential expression of ion channel subunits in human DSM. These exciting new data offer vital clues to the relative importance of the TRPM4 and Kv7 channels in regulating human bladder function. FUNDING: NIH R01 HL-149662 to M. Dennis Leo, NIH P20 DK-123971, NIH R01 DK-106964, and Van Vleet Endowment to Georgi V. Petkov. (See the 2 posters from this P20 Center for more details about this Center’s research) CAIRIBU P20 Exploratory Centers for Interdisciplinary Research in Benign Urology Vanderbilt University Medical Center 2019-2021 PI, Maria Hadjifrangiskou, PhD THE VANDERBILT UROLOGIC INFECTION REPOSITORY, A RESOURCE FOR PERSONALIZED CLINICAL DISCOVERY SUMMARY. In personalized medicine, the care of each patient is guided by his/her unique clinical circumstances. At its foundation, however, this paradigm holds a concurrent need for personalized science, in which technologies are developed and hypothesis explored in light of individual diversity. Critically, this diversity also includes unique microbial populations, which can augment the onset, progression, and treatment of disease. Within the field of benign urology, one of the most common pathologies—urinary tract infections (UTIs)—is also one of the most heterogenous, as the risk factors, symptomatology, and outcomes can vary significantly from patient to patient. Not surprisingly, the complexity of UTIs extends beyond the host, with tremendous genotypic and phenotypic diversity among the species/strains of microbes that e licit these infections. To better align the management of UTIs with the goals of precision care, our understanding of pathophysiology must become more nuanced, as we network in tandem the inherent diversity of host and microbe. To these ends, we propose a resource that provides an interconnected picture of both components, the Vanderbilt Urologic Infection Repository (VUIR). With our institution's unique foundation in medical informatics, we will create a searchable database of clinical parameters from bacteriuric patients (many thousands of cases annually), together with microbiologic data on the organisms.

Glibenclamide blocks ATP‐sensitive K+ channels (Kir6.x‐SUR) via sulphonylurea receptor (SUR) subunit engagement. Glibenclamide can also inhibit TRPM4‐SUR channel complexes via SUR interaction or TRPM4 channels directly. Recently, our group has found that glibenclamide inhibits whole‐cell non‐selective cation (NSC) currents and spontaneous phasic contractions of guinea pig urinary bladder smooth muscle (UBSM) cells and isolated tissue strips, respectively, although less effectively than 9‐phenanthrol, a TRPM4 channel inhibitor. Here, the age‐dependent effects of glibenclamide and 9‐phenanthrol were determined on guinea pig UBSM whole‐cell NSC currents and spontaneous phasic contractions. Urinary bladders were obtained from young (5–8 wk old, 350–650 g) and retired breeder (RB, 0.5–1.5 yr old, >800 g) male Hartley guinea pigs (Charles River). Mucosa was removed and UBSM strips prepared for isometric tension recordings (spontaneous phasic contractions) or for subsequent enzymatic treatment yielding freshly isolated UBSM cells. UBSM cells were then used in amphotericin‐perforated patch‐clamp recordings either (1) whole‐cell NSC currents optimized by blocking K+ and Ca2+ selective currents or (2) cell membrane potential (I=0) with K+ and Ca2+ channel currents intact. Glibenclamide (100 μM) and 9‐phenanthrol (100 μM) inhibited the voltage‐step‐induced NSC currents in RB‐group UBSM cells; at +106 mV, the respective decreases were 36.6±4.8% (n=6, p<0.0001) and 42.9±3.9% (n=9, p<0.001). In UBSM cells of young guinea pigs, 9‐phenanthrol (100 μM, at +106 mV, 50.4±2.9%, n=7, p<0.001) — but not glibenclamide (100 μM, at +106 mV, 4.6±8.3%, n=6) — caused attenuation in the whole‐cell NSC currents. Glibenclamide (100 μM) did not change the membrane potential in UBSM cells of either young (control: −27.8±4.0 mV; change: +4.2±2.4 mV, n=9, p=0.11) or RB (control: −11.6±4.0 mV; change: −0.42±0.8 mV, n=4, p=0.65) guinea pigs. Of note, a majority of UBSM cells of young (5/9 cells), but not RB‐group (0/4 cells), responded to glibenclamide with depolarization (>3 mV). Spontaneous phasic contractions were concentration‐dependently inhibited by glibenclamide and 9‐phenanthrol in UBSM strips of both age groups (n=5–10). Only glibenclamide displayed age‐dependent differential effects on the muscle force (ANOVA, p=0.04), and the phasic contraction amplitude just failed to reach the statistical significance (p=0.07). IC50 values of glibenclamide for the two parameters were 2–3‐fold more potent in young‐ (~14, 15 μM) than RB‐group UBSM strips (31, 45 μM) with high maximum inhibitions (72–93%, n=7–10). This supports the concept that in RB guinea pigs glibenclamide‐sensitive NSC currents oppose UBSM relaxation. In summary, glibenclamide but not 9‐phenanthrol exhibits age‐dependent effects on whole‐cell NSC currents and spontaneous phasic contractions. In old but not young guinea pigs, glibenclamide‐sensitive NSC channels may contribute to the regulation of UBSM excitability and contractility.Support or Funding InformationNIH R01‐DK106964 and P20‐DK123971 to Georgi V. Petkov.

Relaxation and contraction of the urinary bladder smooth muscle, also known as the detrusor smooth muscle (DSM), facilitate the micturition cycle. DSM contractility depends on cell excitability, which is established by the synchronized activity of multiple diverse ion channels. K+ channels, the largest family of channels, control DSM excitability by maintaining the resting membrane potential and shaping the action potentials that cause the phasic contractions. Among the members of the voltage-gated K+ (Kv) channel superfamily, Kv type 7 (Kv7) channels — Kv7.1-Kv7.5 members encoded by KCNQ1-KCNQ5 genes — have been recently identified as functional regulators in various cell types including vascular, cardiac, and neuronal cells. Their regulatory roles in DSM, however, are just now emerging and remain to be elucidated. To address this gap, our research group has initiated the systematic investigation of human DSM Kv7 channels in collaboration with clinical urologists. In this comprehensive review, we summarize the current understanding of DSM Kv7 channels and highlight recent discoveries in the field. We describe Kv7 channel expression profiles at the mRNA and protein levels, and further elaborate on functional effects of Kv7 channel selective modulators on DSM excitability, contractility, and intracellular Ca2+ dynamics in animal species along with in vivo studies and the limited data on human DSM. Within each topic, we highlight the main observations, current gaps in knowledge, and most pressing questions and concepts in need of resolution. We emphasize the lack of systematic studies on human DSM Kv7 channels that are now actively ongoing in our laboratory.

Urinary bladder smooth muscle (UBSM), also known as detrusor smooth muscle, forms the bladder wall and ultimately determines the two main attributes of the organ: urine storage and voiding. The two functions are facilitated by UBSM relaxation and contraction, respectively, which depend on UBSM excitability shaped by multiple ion channels. In this review, we summarize the current understanding of key ion channels establishing and regulating UBSM excitability and contractility. They include excitation-enhancing voltage-gated Ca 2+ (Ca v ) and transient receptor potential channels, excitation-reducing K + channels, and still poorly understood Cl − channels. Dynamic interplay among UBSM ion channels determines the overall level of Ca v channel activity. The net Ca 2+ influx via Ca v channels increases global intracellular Ca 2+ concentration, which subsequently triggers UBSM contractility. Here, for each ion channel type, we describe UBSM tissue/cell expression (mRNA and protein) profiles and their role in regulating excitability and contractility of UBSM in various animal species, including the mouse, rat, and guinea pig, and, most importantly, humans. The currently available data reveal certain interspecies differences, which complicate the translational value of published animal research results to humans. This review highlights recent developments, findings on genetic knockout models, pharmacological data, reports on UBSM ion channel dysfunction in animal bladder disease models, and the very limited human studies currently available. Among all gaps in present-day knowledge, the unknowns on expression and functional roles for ion channels determined directly in human UBSM tissues and cells under both normal and disease conditions remain key hurdles in the field.

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 (Mcoln1-/- ) 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 Mcoln1-/- 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 Mcoln1-/- mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1-/- mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.Copyright © 2020 the Author(s). Published by PNAS. PMID: 33199609 Funding information This work was supported by: NHLBI NIH HHS, United States Grant ID: R01 HL146054

Ion channels of the urinary bladder smooth muscle (UBSM) determine cellular excitability and contractility and, hence, regulate the two main functions of the organ, urine storage and voiding. Preparation of the viable, fresh, and single UBSM cells presents an important technical challenge for subsequent biophysical and electrophysiological studies. Here, we describe a method for successfully obtaining freshly-isolated human single UBSM cells from patient-donors undergoing open bladder surgeries and its application for the recording of voltage-step-induced cation currents. The initial step in the method involves dissection to separate the UBSM layer (also known as detrusor smooth muscle and muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa). The UBSM is then cut into small pieces in Ca2+-free (nominal) dissection/digestion solution (DS). UBSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ∼37 °C for 30-45 min per step. Following washes with enzyme-free DS and trituration with a fire-polished pipette, the pieces release single UBSM cells. Freshly-isolated UBSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations. Specifically, single UBSM cells contract in response to the muscarinic agonist carbachol (3 μM). In amphotericin-B perforated patch-clamp recordings, the TRPM4 channel blocker 9-phenanthrol concentration-dependently reduces voltage-step-induced cation currents. UBSM cells can also be studied by other techniques such as: single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. Studies on freshly-isolated human UBSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable channels in the urinary bladder and will continue as a gold standard in elucidating UBSM cellular properties and regulatory mechanisms. Funding: NIH R01DK106964 grant to Georgi V. Petkov.

Cl − channels serve as key regulators of excitability and contractility in vascular, intestinal, and airway smooth muscle cells. We recently reported a Cl − conductance in detrusor smooth muscle (DSM) cells. Here, we used the whole cell patch-clamp technique to further characterize biophysical properties and physiological regulators of the Cl − current in freshly isolated guinea pig DSM cells. The Cl − current demonstrated outward rectification arising from voltage-dependent gating of Cl − channels rather than the Cl − transmembrane gradient. An exposure of DSM cells to hypotonic extracellular solution (Δ 165 mOsm challenge) did not increase the Cl − current providing strong evidence that volume-regulated anion channels do not contribute to the Cl − current in DSM cells. The Cl − current was monotonically dependent on extracellular pH, larger and lower in magnitude at acidic (5.0) and basic pH (8.5) values, respectively. Additionally, intracellularly applied phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] analog [PI(4,5)P 2 -diC8] increased the average Cl − current density by approximately threefold in a voltage-independent manner. The magnitude of the DSM whole cell Cl − current did not depend on the cell surface area (cell capacitance) regardless of the presence or absence of PI(4,5)P 2 -diC8, an intriguing finding that underscores the complex nature of Cl − channel expression and function in DSM cells. Removal of both extracellular Ca 2+ and Mg 2+ did not affect the DSM whole cell Cl − current, whereas Gd 3+ (1 mM) potentiated the current. Collectively, our recent and present findings strongly suggest that Cl − channels are critical regulators of DSM excitability and are regulated by extracellular pH, Gd 3+ , and PI(4,5)P 2 .

Interstitial cells of Cajal (ICC) generate electrical pacemaker activity in the gastrointestinal (GI) tract known as slow waves, which regulate GI motility. ICC express both the Kit receptor tyrosine kinase protein and a Ca 2+ -activated Cl - -channel, encoded by the anoctamin1 (Ano1) protein, which is an essential contributor to the Ca 2+ cycling of ICC and slow wave pacemaking. Recent dye-loading imaging studies have demonstrated Ca 2+ transients in ICC in isolated tissue preparations. The main aim of this study was to develop a method that allows Ca 2+ transients to be registered to structural ICC network data. Confocal image stacks of ICC labeled for Kit or Ano1 and Ca 2+ recording data were processed using a thresholding protocol. The Ca 2+ transients were then registered to the ICC structural network. First, a general idea of the placement was found by mapping the field-of-view of the Ca 2+ transient data to the distorted tissue that contained the ICC network image. The errors in the registration were then corrected for by warping the internal Ca 2+ transient field according to the structural network. In data sets from tissues with induced, targeted knockdown of Ano1 expression in a subset of ICC, agreement between the Ca 2+ transient data and structural network was 68 ± 10%. This level of agreement allowed selective extraction of Ca 2+ data from Ano1-positive (Ano1+) and Ano1-negative (Ano1-) ICC. In the future, this technique will allow investigation into the functional properties of ICC in relation to the level of knockdown of specific ICC associated proteins.