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Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function

ABSTRACT We recently reported key physiologic roles for Ca2+-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca2+-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca2+, inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the sarcoplasmic reticulum represents a potential Ca2+ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP3Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca2+ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca2+-mediated TRPM4-IP3R regulatory mechanism. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP3Rs with the selective IP3R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP3Rs have a key role in mediating the Ca2+-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM.


Introduction
The functions of the urinary bladder-the principle organ responsible for the storage and periodic release of urine-are mediated by the activity of detrusor smooth muscle (DSM). In DSM cells, it is well-established that Ca 2C influx through L-type voltage-gated Ca 2C channels underlies the depolarization phase of spontaneous action potentials while the large-conductance voltage-and Ca 2C -activated K C (BK) channels, in addition to other voltage-gated K V channels, work in opposition to L-type voltage-gated Ca 2C channel activity to promote membrane hyperpolarization. [1][2][3][4] Albeit these mechanisms have been known for many years in DSM, recent studies from our group have identified an important role for an additional Ca 2C -sensitive cationic conductance regulating DSM function in rodents and humans. [5][6][7][8] Indeed, Ca 2C -activated monovalent cation channels known as the transient receptor potential (TRP) melastatin 4 (TRPM4) channels are functionally expressed in rat, guinea pig, and human DSM. [5][6][7][8] Of the 28 members that constitute the TRP channel superfamily, TRPM4 channels, in addition to TRPM5, are the only TRP channel members selective for monovalent cations (K C and Na C ) over Ca 2C . 9,10 TRPM4 channel functional roles in excitable cells and tissues, including rodent [6][7][8] and human DSM, [5][6][7][8] have begun to emerge in recent years following studies using the novel and selective TRMP4 channel inhibitor, 9-phenanthrol. [6][7][8][11][12][13] We recently provided the first detailed study demonstrating the expression and key functional roles for TRPM4 channels in human DSM excitability and contractility using the selective TRPM4 channel inhibitor 9-phenanthrol. 5 Our novel study was the central topic of an editorial focus in the American Journal of Physiology -Cell Physiology, 14 which highlighted the attractiveness of the TRPM4 channels as potential novel targets for the pharmacological control of the urinary bladder. Under pathophysiological conditions, DSM dysfunction is associated with some forms of overactive bladder. 15 The TRPM4 channels, in addition to the cellular mechanisms regulating their activity, represent an exciting and novel therapeutic approach to the treatment of various bladder disorders and the collateral effects these conditions inflict on patient quality of life. Nonetheless, significant work still remains to ascertain the cellular mechanisms regulating TRPM4 channel activity in human DSM function to further validate their potential therapeutic role for bladder disorders.
It has been suggested that Ca 2C release from the sarcoplasmic reticulum (SR) inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) may constitute a primary Ca 2C source for TRPM4 channel activation. 16 The concept of IP 3 R-mediated Ca 2C release serving as critically important Ca 2C source for TRPM4 channel activation, as opposed to Ca 2C influx through L-type voltage-gated Ca 2C channels, was supported by the observation that removal of extracellular Ca 2C had no acute effects on TRPM4 channel activity. 16 Whether this TRPM4-IP 3 R regulatory mechanism exists in human DSM, however, is completely unknown. Therefore, the current study sought to investigate the molecular and functional interaction of the TRPM4 channels and IP 3 Rs in human DSM. This was achieved via a combined experimental approach using in situ proximity ligation assays (PLA) and perforated patch-clamp electrophysiology in combination with pharmacological tools. This study reveals the novel mechanism whereby SR IP 3 Rs regulate the excitability of human DSM through molecular and functional interactions with TRPM4 channels.

Human DSM tissue acquisition
Human DSM tissues were acquired from donor patients undergoing routine open bladder surgeries as described previously. 5,17,18 All procedures attendant upon the collection of human DSM tissues have been reviewed and approved by the Institutional Review Board of the Medical University of South Carolina (protocol number Pro00045232). 6 Caucasian patients (3 men and 3 women; average age 64.5 § 11.2 years) without preoperative histories of overactive bladder and American Urological Association symptom scores <8 were used for this study. Following surgeries, human DSM tissues were stored on ice in a container containing HEPES-buffered dissection solution (see Solutions and Drugs) for immediate transport to the laboratory for DSM cell isolation.

DSM cell isolation
Human DSM single cells were enzymatically isolated using a combination of papain and collagenase as described previously. 5,17,18 Freshly-isolated DSM cells were used for in situ PLA or electrophysiological experiments within 12 h following isolation.
In situ proximity ligation assay (PLA) In situ PLA was performed on freshly-isolated DSM cells using the Duolink Ò in situ orange starter kit goat/rabbit (Cat. No: DUO92106, Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. DSM cells underwent fixation in 2% paraformaldehyde (10 min, 37 C) followed by 2 15-min washes in phosphate buffered saline. DSM cells were blocked in Duolink blocking solution (30 min, 37 C). Subsequently, DSM cells were incubated with the anti-rabbit IP 3 R and goat anti-TRPM4 channel antibodies (Santa Cruz Biotechnology, Dallas, TX; catalog numbers SC-27540 TRPM4; SC-28614 IP 3 R), respectively, in Duolink antibody diluent solution overnight at 4 C, followed by 2 £ 5 min washes in Duolink 1x wash buffer A solution. The rabbit anti-K v 7.5 antibody was also obtained from Santa Cruz (catalog number SC-50416). DSM cells were then incubated with the Duolink PLA probe anti-rabbit MINUS and anti-goat PLUS oligonucleotide-conjugated secondary antibodies (1 h, 37 C), followed by 2 £ 5 min washes in Duolink 1x wash buffer A solution. DSM cells then underwent incubation in ligase-ligation solution (30 min, 37 C), which hybridizes the 2 PLA MINUS and PLUS probes when in close proximity (< 40 nm). Following ligation, DSM cells were washed 2 £ 2-min in Duolink 1x wash buffer A solution. Finally, DSM cells were incubated in amplification-polymerase solution (100 min, 37 C) containing nucleotides and fluorescently-labeled oligonucleotides. The oligonucleotide arm of one PLA probe is a primer for rolling-circle amplification. The fluorescently-labeled oligonucleotides hybridize to the rolling-circle amplification product thus permitting detection. Following amplification, DSM cells were washed 2 £ 10 min in 1x wash buffer B and then a 1 min wash in 0.01x wash buffer B. Slides were then mounted using minimal volume of Duolink in situ mounting medium with 4',6-diamidino-2-phenylindole (DAPI). PLA signals (Duolink In Situ Detection Reagents Orange (λ excitation/emission D 554/576 nm)) were detected using laser scanning LSM 700 confocal microscope (Carl Zeiss, Germany) with a 63x oil immersion objective.

Patch-clamp electrophysiology
The amphotericin B-perforated whole cell patchclamp technique was used in all of the electrophysiological recordings as described previously. 5,7,8,19 At the holding potential of ¡70 mV (corrected for junction potential), the total open channel probability (NP o ) of TRPM4 channel-mediated transient inward cation currents (TICCs) before and after the addition of xestospongin-C was analyzed. A stable recording period of at least 5-10 min before and after the addition of xestospongin-C was used for analysis with pCLAMP version 10.2 software (Molecular Devices, Union City, CA).

Data analysis and statistics
The TICCs were analyzed as open channel probability (NP o ) as described previously. 5 Data are summarized as means § SEM for n (the number of DSM cells) isolated from N (the number of patients). Data were compared using a paired Student's t-test. A P value <0.05 was considered statistically significant.

TRPM4 channels are colocalized with the IP 3 Rs in human DSM cells
In order for the discrete Ca 2C release events from SR IP 3 Rs to regulate TRPM4 channel activity in human DSM, the IP 3 Rs and TRPM4 channels must be located within close apposition. To determine TRPM4 channel colocalization with IP 3 Rs, we conducted Duolink in situ PLA on freshly-isolated human DSM cells. To elucidate TRPM4/IP 3 R colocalization, human DSM cells were incubated with the anti-TRPM4 goat and anti-IP 3 R rabbit antibodies, respectively. As shown in Fig. 1A, co-incubation of TRPM4 and IP 3 R antibodies positively confirmed that TRPM4 channels and IP 3 Rs are located in close proximity (< 40 nm) to the cell membrane as indicated by the bright red fluorescence. Fluorescent detection was not observed when the anti-TRPM4 goat antibody was omitted (Fig. 1B). As a negative control, we used a rabbit antibody specific for the voltage-gated K V 7.5 channel subtype, which is expressed in human DSM, 20,21 but not expected to interact with TRPM4 channels. Incubation of human DSM cells with anti-TRPM4 and anti-K V 7.5 rabbit antibodies, respectively, resulted in no observable fluorescent signal (Fig. 1). Results were confirmed in at least 3 human DSM cells.

IP 3 R pharmacological inhibition with xestospongin-C decreases transient inward cation current activity in human DSM cells
In non-DSM cells, TRPM4 channels have been shown to be regulated by Ca 2C release events originating from the SR IP 3 Rs. 22 Therefore, SR IP 3 R Ca 2C release activity may constitute a fundamentally important cellular mechanism regulating TRPM4 channel activity in human DSM. To ascertain whether SR IP 3 Rs potentially regulate TRPM4 channel activity in human DSM, we examined the physiologic consequences of IP 3 R pharmacological inhibition on TRPM4 channel-mediated TICCs. At the holding potential of -70 mV, where BK channel activity is negligible, 8 pharmacological inhibition of the IP 3 Rs with the selective inhibitor xestospongin-C (1 mM) significantly reduced TICC activity (NP o ) from 2.0 § 1.1 in control conditions to 1.3 § 1.0 in the presence of 1 mM xestospongin-C (n D 6, N D 4; P<0.05; Fig. 2). These results support the concept that Ca 2C release events from SR IP 3 Rs are involved in the regulation of TRPM4 channel-mediated TICCs in human DSM cells (Fig. 3).

Discussion
This study sought to determine whether IP 3 Rs of the SR regulate TRPM4 channel activity in human DSM function. This was achieved by elucidating IP 3 R-TRPM4 channel spatial and functional interactions in human DSM by using in situ PLA and perforated patch-clamp electrophysiology in combination with the IP 3 R inhibitor xestospongin-C. In situ PLA confirmed co-localization between IP 3 Rs and TRPM4 channels in human DSM isolated cells. Further, IP 3 R pharmacological inhibition with xestospongin-C led to the attenuation of TICCs. This study established the novel finding that TRPM4 channels and SR IP 3 Rs are physically and functionally coupled in the regulation of human DSM excitability.
Several studies from our laboratory have revealed key roles for the TRPM4 channels in regulating the excitability and contractility of DSM in rodents. [6][7][8] More recently, we further explored the translational implications of our findings in rodents by investigating the physiologic roles for the TRPM4 channels in clinically-characterized human DSM. 5 As summarized in a recent editorial article in the American Journal of Physiology Cell Physiology, 14 our group revealed critical roles for the TRPM4 channels in regulating DSM function. TRPM4 channel expression is abundant in human DSM and species-related differences in the functional roles of the TRPM4 channels have been reported between human and rodents. 5 This previous report by our group provided compelling evidence at the cellular level in human DSM demonstrating the TRPM4 channel inhibitor 9-phenanthrol had a substantially greater inhibitory effect on DSM cell excitability in human versus rodent DSM cells. 5 Utilizing the selective TRPM4 channel inhibitor 9phenanthrol, we reported that the pharmacological inhibition of the TRPM4 channels hyperpolarized the membrane potential in human DSM cells and caused inhibition of spontaneous phasic, pharmacologicallyinduced, and nerve-evoked contractions in human DSM isolated strips. 5 Among the mechanisms underlying the inhibitory effects of 9-phenanthrol on human DSM excitability and contractility includes the attenuation of TICCs. [5][6][7][8] TICCs are caused by Na C influx following Ca 2C -dependent TRPM4 channel activation which, under physiologic conditions, functions to enhance DSM cell excitability. 5,23,24 As TRPM4 channels are activated by Ca 2C , it was critically important to determine the specific Ca 2C -dependent molecular and cellular mechanisms involved in the regulation of TRPM4 channel activity. Albeit well-known, there are 2 key sources for the elevation of intracellular Ca 2C concentrations, including: 1) Ca 2C entry through L-type voltage-gated Ca 2C channels at the cell membrane, and 2) Ca 2C release events through SR ryanodine receptors or IP 3 Rs. 4,24 In particular,  . Illustration of the IP 3 R regulatory role of TRPM4 channels in human DSM function. TRPM4 channels conduct monovalent cations (primarily Na C under resting conditions) and control human DSM membrane potential. TRPM4 channels are controlled by IP 3 Rmediated Ca 2C release from the sarcoplasmic reticulum as inhibition of the IP 3 Rs with xestospongin-C reduces TRPM4 channel activity. Pharmacological blockade of TRPM4 channels with 9-phenanthrol hyperpolarizes the DSM cell membrane, inhibits the voltage-gated Ca 2C channels, reduces intracellular Ca 2C levels and promotes DSM relaxation. 5 emerging evidence from non-DSM cell types has shown that members of the TRP channel family, including TRPM4, can be activated by intracellular Ca 2C released from SR IP 3 Rs. IP 3 R-mediated activation of adjacent TRPM4 channels, as has been reported in non-DSM cell types, 16,25 would lead to depolarization of the cell membrane potential, activation of L-type voltage-gated Ca 2C channels, an increase in global intracellular Ca 2C concentrations, and thus the activation of DSM contractions. In fact, in cerebral artery myocytes, sustained TRPM4 channel activity was shown to be dependent on Ca 2C released from the SR IP 3 Rs. 26 Given the critically important role for the TRPM4 channels in human DSM excitability and contractility, we sought to examine whether IP 3 Rs and TRPM4 channels functionally interact to regulate human DSM function. Our results demonstrated that xestospongin-C, a selective IP 3 R inhibitor, significantly decreased TICC activity in human DSM cells (Fig. 2). These results are consistent with IP 3 R-dependent regulation of TRPM4 channels in human DSM by a similar mechanism as was suggested for cerebral artery myocytes. Ca 2C released from the SR IP 3 Rs are localized and transient events as has been demonstrated in murine colonic myocytes and rabbit portal vein myocytes. 12,27 Hence, localized Ca 2C release events for the SR IP 3 Rs must be in close apposition to the plasmalemmal TRPM4 channels in order for IP 3 R-TRPM4 channel functional coupling. Thus, to ascertain the potential co-localization for the TRPM4 channels and IP 3 Rs, we used the Duolink in situ PLA technique in combination with TRPM4 channel and IP 3 R-specific antibodies. As illustrated by Fig. 1, in situ PLA experiments demonstrated the co-localization of IP 3 Rs and TRPM4 channels in freshly-isolated human DSM cells within the vicinity of the cell membrane. These results are in contrast to findings from arterial myocytes, where IP 3 R co-localization was confirmed with TRPC3 channels, while no co-localization with either the TRPM4 or TRPC6 channels was found. 28 The existence of IP 3 R co-localization with TRPM4 channels in DSM, but not in arterial myocytes, may suggest TRPM4 channel-IP 3 R spatial interactions follow, to an extent, a certain level of tissue-specific expression.
While our current data provide strong evidence to support the spatial and functional coupling of the IP 3 Rs and TRPM4 channels in human DSM (Figs. 1-2), we cannot exclude potential involvement of alternative signaling pathways. IP 3 and Ca 2C are the 2 central modulators of SR IP 3 R activity. 29 Indeed, IP 3 R activity has been shown to be regulated by IP 3 generated downstream from the activation of G q/11 -coupled muscarinic receptor pathways involving phospholipase C hydrolysis of phosphatidylinositol bisphosphate (PIP 2 ). 29 Further, more recent studies have shown Ca 2C -permeable TRPC6 channels are spatially and functionally coupled with the SR IP 3 Rs in cerebral arterial myocytes. 30 Thus, TRPC6 activation of the IP 3 Rs leads to Ca 2C release events from the SR IP 3 Rs, which then activate adjacent TRPM4 channels at the cell membrane. 30 Also, in the vasculature, protein kinase C was shown to modulate vascular myogenic tone in a TRPM4 channel-dependent manner. 31,32 Consistently, in a separate study on HEK-293 cells expressing recombinant TRPM4 channels, the sensitivity for TRPM4 channels to Ca 2C -dependent activation is enhanced by PKC phosphorylation. 33 Whether these potential TRPM4 channel regulatory mechanisms exist in human DSM is yet to be explored. Our data confirm IP 3 R-TRPM4 channel functional coupling, and this work represents a foundational study in the human urinary bladder and provides a solid platform for subsequent mechanistic studies in human DSM (Fig. 3).
The key functional role of the TRPM4 channels in promoting DSM excitability has provided an intriguing opportunity for potential therapeutic exploitation in the context of overactive bladder syndrome. Specifically, pharmacological modulators of the TRPM4 channels or mechanisms controlling their activity may represent a promising novel approach for treatment of bladder dysfunction. Given the critical role of the TRPM4 channels in human DSM function and dysfunction, further efforts to ascertain TRPM4 channel regulatory mechanisms are critical for confirming and validating their potential therapeutic utility. The current study has provided novel mechanistic insight into TRPM4 channel regulation by SR IP 3 Rs in human DSM. As current treatments of overactive bladder lack desired efficacy, research efforts to identify novel therapeutic modalities are urgently needed and examining TRPM4 channel physiologic roles and mechanisms of regulation is of scientific and clinical relevance.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.