Small-conductance, Ca²⁺-activated K⁺ channel 2 is the key functional component of SK channels in mouse urinary bladder

Abstract

-activated K ϩ (SK) channels play an important role in regulating the frequency and in shaping urinary bladder smooth muscle (UBSM) action potentials, thereby modulating contractility. Here we investigated a role for the SK2 member of the SK family (SK1-3) utilizing: 1) mice expressing ␤-galactosidase (␤-gal) under the direction of the SK2 promoter (SK2 ␤-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2 Ϫ/Ϫ mice) to assess SK2 function. In SK2 ␤-gal mice, UBSM staining was observed, but staining was undetected in the urothelium. Consistent with this, urothelial SK2 mRNA was determined to be 4% of that in UBSM. Spontaneous phasic contractions in wild-type (SK2 ϩ/ϩ ) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC 50 ϭ 0.16 nM), whereas phasic contractions of SK2 Ϫ/Ϫ strips were unaffected. Nerve-mediated contractions of SK2 ϩ/ϩ UBSM strips were also increased by apamin, an effect absent in SK2 Ϫ/Ϫ strips. Apamin increased the sensitivity of SK2 ϩ/ϩ UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 Ϯ 4 Hz, n ϭ 6; apamin, 12 Ϯ 2 Hz, n ϭ 7; P Ͻ 0.05). In contrast, the sensitivity of SK2 Ϫ/Ϫ UBSM strips was unaffected by apamin. Here we provide novel insight into the molecular basis of SK channels in the urinary bladder, demonstrating that the SK2 gene is expressed in the bladder and that it is essential for the ability of SK channels to regulate UBSM contractility.bladder; contractility; small-conductance calcium-activated potassium channel; apamin THE COORDINATION OF NEURONAL and smooth muscle electrical activity is critical to the maintenance of proper urinary bladder function. Micturition occurs through the initiation of action potentials within the parasympathetic nerves leading to the bladder. These neuronal action potentials on reaching efferent terminals evoke the release of the excitatory neurotransmitters, acetylcholine and ATP. Acetylcholine and ATP bind to muscarinic (M 2 , M 3 ) and purinergic (P 2X1 ) receptors, respectively, located in the urinary bladder smooth muscle (UBSM) membrane. These transmitters contract the bladder via a coordinated potentiation of UBSM action potentials, which occur "spontaneously" during bladder filling functioning to maintain an appropriate basal bladder tone (10).Overactive bladder, a major underlying cause of urinary incontinence, is frequently caused by alterations in neuronal and/or UBSM electrical activity. Currently, the main therapy used to treat overactive bladder is muscarinic receptor antagonists that function to reduce the coupling of excitatory acetylcholine to UBSM muscarinic receptors. These agents are somewhat effective but have significant side effects, such as dry mouth, and in some cases can lead to incomplete urine voiding during micturition. Therefore, the identification of novel targets to be used for the development of better therapies for overactive bladder and urinar...

Pharmacological Activation of Small Conductance Calcium-Activated Potassium Channels with Naphtho[1,2-d]thiazol-2-ylamine Decreases Guinea Pig Detrusor Smooth Muscle Excitability and Contractility

“…As shown here, the pharmacological activation of the DSM SK channels further facilitates this physiological process. Our findings on the pharmacological activation of the SK channel in DSM are in line with previous studies using conditional and conventional SK2 and SK3 channel knockout mouse models (Herrera et al, 2003;Thorneloe et al, 2008). Selective SK3 channel overexpression reduces DSM spontaneous phasic contraction frequency and increases bladder capacity, whereas selective suppression of SK3 channel expression results in DSM overactivity (Herrera et al, 2003).…”

“…In guinea pig and mouse DSM, SK channel inhibition increases the spontaneous phasic contraction amplitude and decreases the contraction frequency (Herrera et al, 2000(Herrera et al, , 2003Imai et al, 2001;Thorneloe et al, 2008). A recent report indicated that NS4591, an activator of SK/IK channels, inhibits EFS-and carbachol-induced contractions in rat, pig, and human DSM .…”

“…In a genetic mouse model, SK3 channel overexpression elevates SK currents and decreases the frequency of DSM phasic contractions in vitro, whereas SK3 channel expression suppression substantially increases nonvoiding contractions in vivo (Herrera et al, 2003). Likewise, studies on SK2 knockout mice suggest that the SK2 channel isoform is another essential contributor to DSM function in the mouse bladder (Thorneloe et al, 2008).…”

A Positive Modulator of KCa2 and KCa3 Channels, 4,5-Dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), Inhibits Bladder Afferent Firing in Vitro and Bladder Overactivity in Vivo

“…A recent study demonstrated the expression of IK as well SK1, SK2, and SK3 in rat DRG neurons, including in putative nociceptive neurons (Mongan et al, 2005). Studies on mice with altered levels of SK2/SK3 expression have supported a role for both SK2 and SK3 in bladder control Thorneloe et al, 2008). However, in these mouse studies SK2 was shown to be expressed in smooth muscle cells, but not in the urothelium, whereas SK3 expression was observed in bladder smooth muscle and urothelium, but neither in afferent nor efferent nerves as evaluated by immunohistochemical costainings with neuronal markers.…”

“…In mice with conditionally suppressed SK3 expression , an involvement of SK3, and possibly other SK forms, in mouse bladder contractility was shown in experiments on bladder strips as well as by in vivo cystometry. Later, when SK2-deficient mice were analyzed, an important role for SK2 in regulation of detrusor contractibility was demonstrated (Thorneloe et al, 2008). The SK2 subunit was shown to be required for the apamin-sensitive regulation of bladder smooth muscle; however, both the actual SK channel composition in detrusor smooth muscle cells and possible species differences still remain to be further explored.…”