The TRPM4 channel is a Ca(2+)-activated, monovalent cation-selective channel of the melastatin transient receptor potential (TRPM) family. The TRPM4 channel is implicated in the regulation of many cellular processes including the immune response, insulin secretion, and pressure-induced vasoconstriction of cerebral arteries. However, the expression and function of the TRPM4 channels in detrusor smooth muscle (DSM) have not yet been explored. Here, we provide the first molecular, electrophysiological, and functional evidence for the presence of TRPM4 channels in rat DSM. We detected the expression of TRPM4 channels at mRNA and protein levels in freshly isolated DSM single cells and DSM tissue using RT-PCR, Western blotting, immunohistochemistry, and immunocytochemistry. 9-Hydroxyphenanthrene (9-phenanthrol), a novel selective inhibitor of TRPM4 channels, was used to examine their role in DSM function. In perforated patch-clamp recordings using freshly isolated rat DSM cells, 9-phenanthrol (30 μM) decreased the spontaneous inward current activity at -70 mV. Real-time DSM live-cell Ca(2+) imaging showed that selective inhibition of TRPM4 channels with 9-phenanthrol (30 μM) significantly reduced the intracellular Ca(2+) levels. Isometric DSM tension recordings revealed that 9-phenanthrol (0.1-30 μM) significantly inhibited the amplitude, muscle force integral, and frequency of the spontaneous phasic and pharmacologically induced contractions of rat DSM isolated strips. 9-Phenanthrol also decreased the amplitude and muscle force integral of electrical field stimulation-induced contractions. In conclusion, this is the first study to examine the expression and provide evidence for TRPM4 channels as critical regulators of rat DSM excitability and contractility.

Overactive bladder syndrome is frequently associated with increased detrusor smooth muscle (DSM) contractility. We tested the hypothesis that pharmacological activation of the large-conductance voltage- and Ca2+-activated K+(BK) channel with NS-1619, a selective BK channel opener, reduces the excitability and contractility of human DSM. We used the amphotericin-perforated whole cell patch-clamp technique on freshly isolated human DSM cells, live-cell Ca2+imaging, and isometric DSM tension recordings of human DSM strips obtained from open bladder surgeries. NS-1619 (30 μM) significantly increased the amplitude of the voltage step-induced whole cell BK currents, and this effect was abolished by pretreatment with 200 nM iberiotoxin (IBTX), a selective BK channel inhibitor. In current-clamp mode, NS-1619 (30 μM) significantly hyperpolarized the resting membrane potential, and the hyperpolarization was reversed by IBTX (200 nM). NS-1619 (30 μM) significantly decreased the intracellular Ca2+level in isolated human DSM cells. BK channel activation with NS-1619 (30 μM) significantly inhibited the amplitude, muscle force, frequency, duration, and tone of the spontaneous phasic and pharmacologically induced DSM contractions from human DSM isolated strips. IBTX (200 nM) suppressed the inhibitory effects of NS-1619 on spontaneous contractions. The amplitude of electrical field stimulation (0.5–50 Hz)-induced contractions was significantly reduced by NS-1619 (30 μM). Our data suggest that pharmacological activation of BK channels could represent a novel treatment option to control bladder dysfunction in humans.

Small conductance Ca 2ϩ -activated K ϩ (SK) and intermediate conductance Ca 2ϩ -activated K ϩ (IK) channels are thought to be involved in detrusor smooth muscle (DSM) excitability and contractility. Using naphtho[1,2-d]thiazol-2-ylamine (SKA-31), a novel and highly specific SK/IK channel activator, we investigated whether pharmacological activation of SK/IK channels reduced guinea pig DSM excitability and contractility. We detected the expression of all known isoforms of SK (SK1-SK3) and IK channels at mRNA and protein levels in DSM by singlecell reverse transcription-polymerase chain reaction and Western blot. Using the perforated patch-clamp technique on freshly isolated DSM cells, we observed that SKA-31 (10 M) increased SK currents, which were blocked by apamin (1 M), a selective SK channel inhibitor. In current-clamp mode, SKA-31 (10 M) hyperpolarized the cell resting membrane potential, which was blocked by apamin (1 M) but not by 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 M), a selective IK channel inhibitor. SKA-31 (10 nM-10 M) significantly inhibited the spontaneous phasic contraction amplitude, frequency, duration, and muscle force in DSM isolated strips. The SKA-31 inhibitory effects on DSM contractility were blocked by apamin (1 M) but not by TRAM-34 (1 M), which did not per se significantly affect DSM spontaneous contractility. SK channel activation with SKA-31 reduced contractions evoked by electrical field stimulation. SKA-31 effects were reversible upon washout. In conclusion, SK channels, but not IK channels, mediate SKA-31 effects in guinea pig DSM. Pharmacological activation of SK channels reduces DSM excitability and contractility and therefore may provide a novel therapeutic approach for controlling bladder dysfunction.

Hristov KL, Chen M, Soder RP, Parajuli SP, Cheng Q, Kellett WF, Petkov GV. KV2.1 and electrically silent KV channel subunits control excitability and contractility of guinea pig detrusor smooth muscle. Am J Physiol Cell Physiol 302: C360 -C372, 2012. First published October 12, 2011; doi:10.1152/ajpcell.00303.2010.-Voltage-gated K ϩ (KV) channels are implicated in detrusor smooth muscle (DSM) function. However, little is known about the functional role of the heterotetrameric KV channels in DSM. In this report, we provide molecular, electrophysiological, and functional evidence for the presence of KV2.1 and electrically silent KV channel subunits in guinea pig DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of the homotetrameric KV2.1, KV2.2, and KV4.2 as well as the heterotetrameric KV2.1/6.3 and KV2.1/9.3 channels, was used to examine the role of these KV channels in DSM function. RT-PCR indicated mRNA expression of KV2.1, KV6.2-6.3, KV8.2, and KV9.1-9.3 subunits in isolated DSM cells. KV2.1 protein expression was confirmed by Western blot and immunocytochemistry. Perforated whole cell patchclamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the KV current in freshly isolated DSM cells. ScTx1 (100 nM) did not significantly change the steady-state activation and inactivation curves for KV current. However, ScTx1 (100 nM) decreased the activation time-constant of the KV current at positive voltages. Although our patch-clamp data could not exclude the presence of the homotetrameric KV2.1 channels, the biophysical characteristics of the ScTx1-sensitive current were consistent with the presence of heterotetrameric KV2.1/silent KV channels. Currentclamp recordings showed that ScTx1 (100 nM) did not change the DSM cell resting membrane potential. ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and muscle tone as well as the amplitude of the electrical field stimulationinduced contractions of isolated DSM strips. Collectively, our data revealed that KV2.1-containing channels are important physiological regulators of guinea pig DSM excitability and contractility. urinary bladder; patch clamp; reverse transcriptase-polymerase chain reaction; Western blot; immunocytochemistry; stromatoxin-1 DETRUSOR SMOOTH MUSCLE (DSM), which makes up the bladder wall, relaxes during bladder filling and contracts phasically during voiding (2). The underlying cause of the spontaneous phasic DSM contractions is the spontaneous action potential and corresponding Ca 2ϩ transients (19 -22, 25-27, 47). Initiation of the action potential in guinea pig DSM arises from the opening of L-type voltage-gated Ca 2ϩ channels followed by an increase in the intracellular Ca 2ϩ concentration (25). The repolarization phase of the DSM action potential is initiated by activation of large-conductance Ca 2ϩ -activated K ϩ (BK) channels, voltage-gated K ϩ (K V ) channels (56), and negative feedback Ca 2ϩ -mediated inhibition of the L-type voltage-gated Ca 2ϩ channels (7,11,19,26,35,46,48), whereas sma...