KV2.1 and electrically silent KV channel subunits control excitability and contractility of guinea pig detrusor smooth muscle

Voltage-gated K(+) (K(V)) channels are implicated in detrusor smooth muscle (DSM) function. However, little is known about the functional role of the heterotetrameric K(V) channels in DSM. In this report, we provide molecular, electrophysiological, and functional evidence for the presence of K(V)2.1 and electrically silent K(V) channel subunits in guinea pig DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of the homotetrameric K(V)2.1, K(V)2.2, and K(V)4.2 as well as the heterotetrameric K(V)2.1/6.3 and K(V)2.1/9.3 channels, was used to examine the role of these K(V) channels in DSM function. RT-PCR indicated mRNA expression of K(V)2.1, K(V)6.2-6.3, K(V)8.2, and K(V)9.1-9.3 subunits in isolated DSM cells. K(V)2.1 protein expression was confirmed by Western blot and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the K(V) current in freshly isolated DSM cells. ScTx1 (100 nM) did not significantly change the steady-state activation and inactivation curves for K(V) current. However, ScTx1 (100 nM) decreased the activation time-constant of the K(V) current at positive voltages. Although our patch-clamp data could not exclude the presence of the homotetrameric K(V)2.1 channels, the biophysical characteristics of the ScTx1-sensitive current were consistent with the presence of heterotetrameric K(V)2.1/silent K(V) channels. Current-clamp 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 stimulation-induced contractions of isolated DSM strips. Collectively, our data revealed that K(V)2.1-containing channels are important physiological regulators of guinea pig DSM excitability and contractility.     

Contribution of K(v)2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra

We have characterized the native voltage-dependent K(+) (K(v)) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K(v)2.1 and K(v)2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEK(Kv2.1) and HEK(Kv2.2)). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch-clamp technique with K(+)-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca(2+)-activated K(+) (BK) currents and depolarized to +40 mV for 500 ms to evoke K(v) currents. These were unaffected by margatoxin, κ-dendrotoxin, or α-dendrotoxin (100 nM, n = 3-5) but were blocked by stromatoxin-1 (ScTx, IC(50) ～130 nM), consistent with the idea that the currents were carried through K(v)2 channels. RNA was detected for K(v)2.1, K(v)2.2, and the silent subunit K(v)9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K(v)2 subtypes and K(v)9.3 in isolated RUSMC. HEK(Kv2.1) and HEK(Kv2.2) currents were blocked in a concentration-dependent manner by ScTx, with estimated IC(50) values of ～150 nM (K(v)2.1, n = 5) and 70 nM (K(v)2.2, n = 6). The mean half-maximal voltage (V(1/2)) of inactivation of the USMC K(v) current was -56 ± 3 mV (n = 9). This was similar to the HEK(Kv2.1) current (-55 ± 3 mV, n = 13) but significantly different from the HEK(Kv2.2) currents (-30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that K(v)2.1 channels contribute significantly to the K(v) current in RUSMC.     

Suppression of human detrusor smooth muscle excitability and contractility via pharmacological activation of large conductance Ca2+-activated K+ channels

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 Ca(2+)-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 Ca(2+) 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 Ca(2+) 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.     

Large-conductance voltage- and Ca2+-activated K+ channels regulate human detrusor smooth muscle function

The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel α-, β(1)-, and β(4)-subunits. Western blot and immunocytochemistry confirmed BK channel α, β(1), and β(4) protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patch-clamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K(+) current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin- and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans.     

Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca²⁺-activated K⁺ channel

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ～3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ～2-fold and hyperpolarized the DSM cell resting membrane potential by ～6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca(2+) sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.     

Kv3 channels contribute to the delayed rectifier current in small cultured mouse dorsal root ganglion neurons

Delayed rectifier voltage-gated K(+) (K(V)) channels are important determinants of neuronal excitability. However, the large number of K(V) subunits poses a major challenge to establish the molecular composition of the native neuronal K(+) currents. A large part (～60%) of the delayed rectifier current (I(K)) in small mouse dorsal root ganglion (DRG) neurons has been shown to be carried by both homotetrameric K(V)2.1 and heterotetrameric channels of K(V)2 subunits with silent K(V) subunits (K(V)S), while a contribution of K(V)1 channels has also been demonstrated. Because K(V)3 subunits also generate delayed rectifier currents, we investigated the contribution of K(V)3 subunits to I(K) in small mouse DRG neurons. After stromatoxin (ScTx) pretreatment to block the K(V)2-containing component, application of 1 mM TEA caused significant additional block, indicating that the ScTx-insensitive part of I(K) could include K(V)1, K(V)3, and/or M-current channels (KCNQ2/3). Combining ScTx and dendrotoxin confirmed a relevant contribution of K(V)2 and K(V)2/K(V)S, and K(V)1 subunits to I(K) in small mouse DRG neurons. After application of these toxins, a significant TEA-sensitive current (～19% of total I(K)) remained with biophysical properties that corresponded to those of K(V)3 currents obtained in expression systems. Using RT-PCR, we detected K(V)3.1-3 mRNA in DRG neurons. Furthermore, Western blot and immunocytochemistry using K(V)3.1-specific antibodies confirmed the presence of K(V)3.1 in cultured DRG neurons. These biophysical, pharmacological, and molecular results demonstrate a relevant contribution (～19%) of K(V)3-containing channels to I(K) in small mouse DRG neurons, supporting a substantial role for K(V)3 subunits in these neurons.     

SK but not IK channels regulate human detrusor smooth muscle spontaneous and nerve-evoked contractions

Animal studies suggest that the small (SK) and intermediate (IK) conductance Ca(2+)-activated K(+) channels may contribute to detrusor smooth muscle (DSM) excitability and contractility. However, the ability of SK and IK channels to control DSM spontaneous phasic and nerve-evoked contractions in human DSM remains unclear. We first investigated SK and IK channels molecular expression in native human DSM and further assessed their functional role using isometric DSM tension recordings and SK/IK channel-selective inhibitors. Quantitative PCR experiments revealed that SK3 channel mRNA expression in isolated DSM single cells was ～12- to 44-fold higher than SK1, SK2, and IK channels. RT-PCR studies at the single-cell level detected mRNA messages for SK3 channels but not SK1, SK2, and IK channels. Western blot and immunohistochemistry analysis further confirmed protein expression for the SK3 channel and lack of detectable protein expression for IK channel in whole DSM tissue. Apamin (1 μM), a selective SK channel inhibitor, significantly increased the spontaneous phasic contraction amplitude, muscle force integral, phasic contraction duration, and muscle tone of human DSM isolated strips. Apamin (1 μM) also increased the amplitude of human DSM electrical field stimulation (EFS)-induced contractions. However, TRAM-34 (1 μM), a selective IK channel inhibitor, had no effect on the spontaneous phasic and EFS-induced DSM contractions suggesting a lack of IK channel functional role in human DSM. In summary, our molecular and functional studies revealed that the SK, particularly the SK3 subtype, but not IK channels are expressed and regulate the spontaneous and nerve-evoked contractions in human DSM.     

Stimulation of beta3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca2+-activated K+ channels

We investigated the role of large-conductance Ca(2+)-activated K(+) (BK) channels in beta3-adrenoceptor (beta3-AR)-induced relaxation in rat urinary bladder smooth muscle (UBSM). BRL 37344, a specific beta3-AR agonist, inhibits spontaneous contractions of isolated UBSM strips. SR59230A, a specific beta3-AR antagonist, and H89, a PKA inhibitor, reduced the inhibitory effect of BRL 37344. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration response curves for contraction amplitude, net muscle force, and tone to the right. Freshly dispersed UBSM cells and the perforated mode of the patch-clamp technique were used to determine further the role of beta3-AR stimulation by BRL 37344 on BK channel activity. BRL 37344 increased spontaneous, transient, outward BK current (STOC) frequency by 46.0 +/- 20.1%. In whole cell mode at a holding potential of V(h) = 0 mV, the single BK channel amplitude was 5.17 +/- 0.28 pA, whereas in the presence of BRL 37344, it was 5.55 +/- 0.41 pA. The BK channel open probability was also unchanged. In the presence of ryanodine and nifedipine, the current-voltage relationship in response to depolarization steps in the presence and absence of BRL 37344 was identical. In current-clamp mode, BRL 37344 caused membrane potential hyperpolarization from -26.1 +/- 2.1 mV (control) to -29.0 +/- 2.2 mV. The BRL 37344-induced hyperpolarization was eliminated by application of iberiotoxin, tetraethylammonium or ryanodine. The data indicate that stimulation of beta3-AR relaxes rat UBSM by increasing the BK channel STOC frequency, which causes membrane hyperpolarization and thus relaxation.     

Low levels of K(ATP) channel activation decrease excitability and contractility of urinary bladder

Activation of ATP-sensitive potassium (K(ATP)) channels can regulate smooth muscle function through membrane potential hyperpolarization. A critical issue in understanding the role of K(ATP) channels is the relationship between channel activation and the effect on tissue function. Here, we explored this relationship in urinary bladder smooth muscle (UBSM) from the detrusor by activating K(ATP) channels with the synthetic compounds N-(4-benzoylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (ZD-6169) and levcromakalim. The effects of ZD-6169 and levcromakalim on K(ATP) channel currents in isolated UBSM cells, on action potentials, and on related phasic contractions of isolated UBSM strips were examined. ZD-6169 and levcromakalim at 1.02 and 2.63 microM, respectively, caused half-maximal activation (K1/2) of K(ATP) currents in single UBSM cells (see Heppner TJ, Bonev A, Li JH, Kau ST, and Nelson MT. Pharmacology 53: 170-179, 1996). In contrast, much lower concentrations (K(1/2) = 47 nM for ZD-6169 and K1/2 = 38 nM for levcromakalim) caused inhibition of action potentials and phasic contractions of UBSM. The results suggest that activation of <1% of K(ATP) channels is sufficient to inhibit significantly action potentials and the related phasic contractions.     

Inhibition of voltage-gated K(+) currents by endothelin-1 in human pulmonary arterial myocytes

Recent studies demonstrate that endothelin-1 (ET-1) constricts human pulmonary arteries (PA). In this study, we examined possible mechanisms by which ET-1 might constrict human PA. In smooth muscle cells freshly isolated from these arteries, whole cell patch-clamp techniques were used to examine voltage-gated K(+) (K(V)) currents. K(V) currents were isolated by addition of 100 nM charybdotoxin and were identified by current characteristics and inhibition by 4-aminopyridine (10 mM). ET-1 (10(-8) M) caused significant inhibition of K(V) current. Staurosporine (1 nM), a protein kinase C (PKC) inhibitor, abolished the effect of ET-1. Rings of human intrapulmonary arteries (0.8-2 mm OD) were suspended in tissue baths for isometric tension recording. ET-1-induced contraction was maximal at 10(-8) M, equal to that induced by K(V) channel inhibition with 4-aminopyridine, and attenuated by PKC inhibitors. These data suggest that ET-1 constricts human PA, possibly because of myocyte depolarization via PKC-dependent inhibition of K(V). Our results are consistent with data we reported previously in the rat, suggesting similar mechanisms may be operative in both species.     

Zacopride selectively activates the Kir2.1 channel via a PKA signaling pathway in rat cardiomyocytes

We recently reported that zacopride is a selective inward rectifier potassium current (IK1 ) channel agonist, suppressing ventricular arrhythmias without affecting atrial arrhythmias. The present study aimed to investigate the unique pharmacological properties of zacopride. The whole-cell patch-clamp technique was used to study IK1 currents in rat atrial myocytes and Kir2.x currents in human embryonic kidney (HEK)-293 cells transfected with inward rectifier potassium channel (Kir)2.1, Kir2.2, Kir2.3, or mutated Kir2.1 (at phosphorylation site S425L). Western immunoblots were performed to estimate the relative protein expression levels of Kir2.x in rat atria and ventricles. Results showed that zacopride did not affect the IK1 and transmembrane potential of atrial myocytes. In HEK293 cells, zacopride increased Kir2.1 homomeric channels by 40.7%±9.7% at 50 mV, but did not affect Kir2.2 and Kir2.3 homomeric channels, and Kir2.1-Kir2.2, Kir2.1-Kir2.3 and Kir2.2-Kir2.3 heteromeric channels. Western immunoblots showed that similar levels of Kir2.3 protein were expressed in rat atria and ventricles, but atrial Kir2.1 protein level was only 25% of that measured in the ventricle. In addition, 5-hydroxytryptamine (5-HT) 3 receptor was undetectable, whereas 5-HT 4 receptor was weakly expressed in HEK293 cells. The Kir2.1-activating effect of zacopride in these cells was abolished by inhibition of protein kinase A (PKA), but not PKC or PKG. Furthermore, zacopride did not activate the mutant Kir2.1 channel in HEK293 cells but selectively activated the Kir2.1 homomeric channel via a PKA-dependent pathway, independent to that of the 5-HT receptor.     

Down-regulation of voltage-gated Ca2+ channels by neuronal calcium sensor-1 is beta subunit-specific

Neuronal Ca(2+) sensor protein-1 (NCS-1) is a member of the Ca(2+) binding protein family, with three functional Ca(2+) binding EF-hands and an N-terminal myristoylation site. NCS-1 is expressed in brain and heart during embryonic and postnatal development. In neurons, NCS-1 facilitates neurotransmitter release, but both inhibition and facilitation of the Ca(2+) current amplitude have been reported. In heart, NCS-1 co-immunoprecipitates with K(+) channels and modulates their activity, but the potential effects of NCS-1 on cardiac Ca(2+) channels have not been investigated. To directly assess the effect of NCS-1 on the various types of Ca(2+) channels we have co-expressed NCS-1 in Xenopus oocytes, with Ca(V)1.2, Ca(V)2.1, and Ca(V)2.2 Ca(2+) channels, using various subunit combinations. The major effect of NCS-1 was to decrease Ca(2+) current amplitude, recorded with the three different types of alpha(1) subunit. When expressed with Ca(V)2.1, the depression of Ca(2+) current amplitude induced by NCS-1 was dependent upon the identity of the beta subunit expressed, with no block recorded without beta subunit or with the beta(3) subunit. Current-voltage and inactivation curves were also slightly modified and displayed a different specificity toward the beta subunits. Taken together, these data suggest that NCS-1 is able to modulate cardiac and neuronal voltage-gated Ca(2+) channels in a beta subunit specific manner.     

Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle

When the urinary bladder is full, activation of parasympathetic nerves causes release of neurotransmitters that induce forceful contraction of the detrusor muscle, leading to urine voiding. The roles of ion channels that regulate contractility of urinary bladder smooth muscle (UBSM) in response to activation of parasympathetic nerves are not well known. The present study was designed to characterize the role of large (BK)- and small-conductance (SK) Ca(2+)-activated K(+) (K(Ca)) channels in regulating UBSM contractility in response to physiological levels of nerve stimulation in UBSM strips from mice. Nerve-evoked contractions were induced by electric field stimulation (0.5-50 Hz) in isolated strips of UBSM. BK and SK channel inhibition substantially increased the amplitude of nerve-evoked contractions up to 2.45 +/- 0.12- and 2.99 +/- 0.25-fold, respectively. When both SK and BK channels were inhibited, the combined response was additive. Inhibition of L-type voltage-dependent Ca(2+) channels (VDCCs) in UBSM inhibited nerve-evoked contractions by 92.3 +/- 2.0%. These results suggest that SK and BK channels are part of two distinct negative feedback pathways that limit UBSM contractility in response to nerve stimulation by modulating the activity of VDCCs. Dysfunctional regulation of UBSM contractility by alterations in BK/SK channel expression or function may underlie pathologies such as overactive bladder.     

Regulation of Guinea Pig Detrusor Smooth Muscle Excitability by 17β-Estradiol: The Role of the Large Conductance Voltage- and Ca2+-Activated K+ Channels

Estrogen replacement therapies have been suggested to be beneficial in alleviating symptoms of overactive bladder. However, the precise regulatory mechanisms of estrogen in urinary bladder smooth muscle (UBSM) at the cellular level remain unknown. Large conductance voltage- and Ca²⁺-activated K⁺ (BK) channels, which are key regulators of UBSM function, are suggested to be non-genomic targets of estrogens. This study provides an electrophysiological investigation into the role of UBSM BK channels as direct targets for 17β-estradiol, the principle estrogen in human circulation. Single BK channel recordings on inside-out excised membrane patches and perforated whole cell patch-clamp were applied in combination with the BK channel selective inhibitor paxilline to elucidate the mechanism of regulation of BK channel activity by 17β-estradiol in freshly-isolated guinea pig UBSM cells. 17β-Estradiol (100 nM) significantly increased the amplitude of depolarization-induced whole cell steady-state BK currents and the frequency of spontaneous transient BK currents in freshly-isolated UBSM cells. The increase in whole cell BK currents by 17β-estradiol was eliminated upon blocking BK channels with paxilline. 17β-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17β-estradiol-BK channel interactions. 17β-Estradiol (100 nM) caused a significant hyperpolarization of the membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline. 17β-Estradiol (100 nM) had no effects on L-type voltage-gated Ca²⁺ channel currents recorded under perforated patch-clamp conditions. This study reveals a new regulatory mechanism in the urinary bladder whereby BK channels are directly activated by 17β-estradiol to reduce UBSM cell excitability.     

Regulation of inward rectifier K+ channels by shift of intracellular pH dependence

The mechanistic link between mitochondrial metabolism and inward rectifier K+ channel activity was investigated by studying the effects of a mitochondrial inhibitor, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on inward rectifiers of the Kir2 subfamily expressed in Xenopus oocytes, using two-electrode voltage-clamp, patch-clamp, and intracellular pH recording. FCCP inhibited Kir2.2 and Kir2.3 currents and decreased intracellular pH, but the pH change was too small to account for the inhibitory effect by itself. However, pre-incubation of oocytes with imidazole prevented both the pH decrease and the inhibition of Kir2.2 and Kir2.3 currents by FCCP. The pH dependence of Kir2.2 was shifted to higher pH in membrane patches from FCCP-treated oocytes compared to control oocytes. Therefore, the inhibition of Kir2.2 by FCCP may involve a combination of intracellular acidification and a shift in the intracellular pH dependence of these channels. To investigate the sensitivity of heteromeric channels to FCCP, we studied its effect on currents expressed by heteromeric tandem dimer constructs. While Kir2.1 homomeric channels were insensitive to FCCP, both Kir2.1-Kir2.2 and Kir2.1-Kir2.3 heterotetrameric channels were inhibited. These data support the notion that mitochondrial dysfunction causes inhibition of heteromeric inward rectifier K+ channels. The reduction of inward rectifier K+ channel activity observed in heart failure and ischemia may result from the mitochondrial dysfunction that occurs in these conditions.     

Expression and function of potassium channels in the human placental vasculature

In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, which are important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil, and anandamide. Experiments were performed at 20, 7, and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1, and TASK1 channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1, and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.     

Neurogenic Detrusor Overactivity Is Associated with Decreased Expression and Function of the Large Conductance Voltage- and Ca2+-Activated K+ Channels

Patients suffering from a variety of neurological diseases such as spinal cord injury, Parkinson’s disease, and multiple sclerosis often develop neurogenic detrusor overactivity (NDO), which currently lacks a universally effective therapy. Here, we tested the hypothesis that NDO is associated with changes in detrusor smooth muscle (DSM) large conductance Ca²⁺-activated K⁺ (BK) channel expression and function. DSM tissue samples from 33 patients were obtained during open bladder surgeries. NDO patients were clinically characterized preoperatively with pressure-flow urodynamics demonstrating detrusor overactivity, in the setting of a clinically relevant neurological condition. Control patients did not have overactive bladder and did not have a clinically relevant neurological disease. We conducted quantitative polymerase chain reactions (qPCR), perforated patch-clamp electrophysiology on freshly-isolated DSM cells, and functional studies on DSM contractility. qPCR experiments revealed that DSM samples from NDO patients showed decreased BK channel mRNA expression in comparison to controls. Patch-clamp experiments demonstrated reduced whole cell and transient BK currents (TBKCs) in freshly-isolated DSM cells from NDO patients. Functional studies on DSM contractility showed that spontaneous phasic contractions had a decreased sensitivity to iberiotoxin, a selective BK channel inhibitor, in DSM strips isolated from NDO patients. These results reveal the novel finding that NDO is associated with decreased DSM BK channel expression and function leading to increased DSM excitability and contractility. BK channel openers or BK channel gene transfer could be an alternative strategy to control NDO. Future clinical trials are needed to evaluate the value of BK channel opening drugs or gene therapies for NDO treatment and to identify any possible adverse effects.