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

Small-conductance Ca2+-activated K+ (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 β-galactosidase (β-gal) under the direction of the SK2 promoter (SK2 β-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2−/− mice) to assess SK2 function. In SK2 β-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+/+) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC50 = 0.16 nM), whereas phasic contractions of SK2−/− strips were unaffected. Nerve-mediated contractions of SK2+/+ UBSM strips were also increased by apamin, an effect absent in SK2−/− strips. Apamin increased the sensitivity of SK2+/+ UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 ± 4 Hz, n = 6; apamin, 12 ± 2 Hz, n = 7; P < 0.05). In contrast, the sensitivity of SK2−/− 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.

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Expression and function of the small-conductance Ca2+-activated K+ channel is decreased in urinary bladder smooth muscle cells from female guinea pig with partial bladder outlet obstruction

International Urology and Nephrology ◽

10.1007/s11255-017-1592-0 ◽

2017 ◽

Vol 49 (7) ◽

pp. 1147-1155 ◽

Cited By ~ 5

Author(s):

Ning Li ◽

Honglin Ding ◽

Xiaoning He ◽

Zizheng Li ◽

Yili Liu

Keyword(s):

Smooth Muscle ◽

Urinary Bladder ◽

Bladder Outlet Obstruction ◽

Outlet Obstruction ◽

K Channel ◽

Bladder Smooth Muscle ◽

Partial Bladder Outlet Obstruction ◽

Urinary Bladder Smooth Muscle ◽

And Function ◽

Small Conductance

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Nerve-released acetylcholine contracts urinary bladder smooth muscle by inducing action potentials independently of IP3-mediated calcium release

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00180.2010 ◽

2010 ◽

Vol 299 (3) ◽

pp. R878-R888 ◽

Cited By ~ 19

Author(s):

Bernhard Nausch ◽

Thomas J. Heppner ◽

Mark T. Nelson

Keyword(s):

Smooth Muscle ◽

Sarcoplasmic Reticulum ◽

Urinary Bladder ◽

Action Potentials ◽

Cyclopiazonic Acid ◽

Electrical Field Stimulation ◽

Field Stimulation ◽

Bladder Smooth Muscle ◽

Electrical Field ◽

Urinary Bladder Smooth Muscle

Nerve-released ACh is the main stimulus for contraction of urinary bladder smooth muscle (UBSM). Here, the mechanisms by which ACh contracts UBSM are explored by determining Ca2+ and electrical signals induced by nerve-released ACh. Photolysis of caged inositol 1,4,5-trisphosphate (IP3) evoked Ca2+ release from the sarcoplasmic reticulum. Electrical field stimulation (20 Hz) induced Ca2+ waves within the smooth muscle that were present only during stimulus application. Ca2+ waves were blocked by inhibition of muscarinic ACh receptors (mAChRs) with atropine and depletion of sarcoplasmic reticulum Ca2+ stores with cyclopiazonic acid (CPA), and therefore likely reflect activation of IP3 receptors (IP3Rs). Electrical field stimulation also increased excitability to induce action potentials (APs) that were accompanied by Ca2+ flashes, reflecting Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs) during the action potential. The evoked Ca2+ flashes and APs occurred as a burst with a lag time of ∼1.5 s after onset of stimulation. They were not inhibited by blocking IP3-mediated Ca2+ waves, but by blockers of mAChRs (atropine) and VDCCs (diltiazem). Nerve-evoked contractions of UBSM strips were greatly reduced by blocking VDCCs, but not by preventing IP3-mediated Ca2+ signaling with cyclopiazonic acid or inhibition of PLC with U73122. These results indicate that ACh released from nerve varicosities induces IP3-mediated Ca2+ waves during stimulation; but contrary to expectations, these signals do not appear to participate in contraction. In addition, our data provide compelling evidence that UBSM contractions evoked by nerve-released ACh depend on increased excitability and the resultant Ca2+ entry through VDCCs during APs.

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SK channel-selective opening by SKA-31 induces hyperpolarization and decreases contractility in human urinary bladder smooth muscle

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00363.2012 ◽

2013 ◽

Vol 304 (2) ◽

pp. R155-R163 ◽

Cited By ~ 17

Author(s):

Rupal P. Soder ◽

Shankar P. Parajuli ◽

Kiril L. Hristov ◽

Eric S. Rovner ◽

Georgi V. Petkov

Keyword(s):

Smooth Muscle ◽

Electrical Field Stimulation ◽

Tissue Level ◽

Isometric Tension ◽

Resting Membrane Potential ◽

Sk Channels ◽

Bladder Smooth Muscle ◽

Patch Clamp Technique ◽

Sk Channel ◽

Urinary Bladder Smooth Muscle

Overactive bladder (OAB) is often associated with increased involuntary detrusor smooth muscle (DSM) contractions during the bladder-filling phase. To develop novel therapies for OAB, it is critical to better understand the mechanisms that control DSM excitability and contractility. Recent studies showed that small-conductance Ca2+-activated K+ (SK) channels, SK3 channels, in particular, regulate human DSM function. However, the concept that SK channel-selective pharmacological activation can decrease the excitability and contractility directly in human DSM needs further exploration. Here, we studied the effect of the novel and potent SK channel activator, SKA-31 (or naphtho [1,2- d]thiazol-2-ylamine), on human DSM excitability and contractility at the cellular and tissue level. We used isometric tension recordings on human DSM-isolated strips and the perforated patch-clamp technique on freshly isolated native human DSM cells. SKA-31 significantly decreased spontaneous phasic contractions of DSM-isolated strips. In the presence of the SK channel blocker, apamin, the inhibitory effects of SKA-31 on the DSM spontaneous phasic contractions were significantly reduced. SKA-31 decreased the carbachol- and KCl-induced contractions in human DSM strips. Electrical field stimulation-induced contractions were significantly attenuated in the presence of SKA-31 at all stimulation frequencies (0.5–50 Hz). SKA-31 hyperpolarized the resting membrane potential of human DSM cells. Apamin abolished the hyperpolarizing effect of SKA-31, indicating the involvement of SK channel activation. These results support the concept that pharmacological activation of SK channels with selective openers may represent an attractive new pharmacological approach for decreasing DSM excitability and contractility, thus controlling OAB.

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Voltage-gated K+ channels sensitive to stromatoxin-1 regulate myogenic and neurogenic contractions of rat urinary bladder smooth muscle

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00036.2010 ◽

2010 ◽

Vol 299 (1) ◽

pp. R177-R184 ◽

Cited By ~ 20

Author(s):

Muyan Chen ◽

Whitney F. Kellett ◽

Georgi V. Petkov

Keyword(s):

Smooth Muscle ◽

Urinary Bladder ◽

Electrical Field Stimulation ◽

Resting Membrane Potential ◽

Bladder Smooth Muscle ◽

Membrane Excitability ◽

Molecular Fingerprints ◽

Rat Urinary Bladder ◽

Voltage Gated ◽

Urinary Bladder Smooth Muscle

Members of the voltage-gated K+ (KV) channel family are suggested to control the resting membrane potential and the repolarization phase of the action potential in urinary bladder smooth muscle (UBSM). Recent studies report that stromatoxin-1, a peptide isolated from tarantulas, selectively inhibits KV2.1, KV2.2, KV4.2, and KV2.1/9.3 channels. The objective of this study was to investigate whether KV channels sensitive to stromatoxin-1 participate in the regulation of rat UBSM contractility and to identify their molecular fingerprints. Stromatoxin-1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and tone in isolated UBSM strips. However, stromatoxin-1 (100 nM) had no effect on the UBSM contractions induced by depolarizing agents such as KCl (20 mM) or carbachol (1 μM). This indicates that, under conditions of sustained membrane depolarization, the KV channels sensitive to stromatoxin-1 have no further contribution to the membrane excitability and contractility. Stromatoxin-1 (100 nM) increased the amplitude of the electrical field stimulation-induced contractions, suggesting also a role for these channels in neurogenic contractions. RT-PCR experiments on freshly isolated UBSM cells showed mRNA expression of KV2.1, KV2.2, and KV9.3, but not KV4.2 channel subunits. Protein expression of KV2.1 and KV2.2 channels was detected using Western blot and was further confirmed by immunocytochemical detection in freshly isolated UBSM cells. These novel findings indicate that KV2.1 and KV2.2, but not KV4.2, channel subunits are expressed in rat UBSM and play a key role in opposing both myogenic and neurogenic UBSM contractions.

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Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.2000.279.1.r60 ◽

2000 ◽

Vol 279 (1) ◽

pp. R60-R68 ◽

Cited By ~ 113

Author(s):

Gerald M. Herrera ◽

Thomas J. Heppner ◽

Mark T. Nelson

Keyword(s):

Smooth Muscle ◽

Urinary Bladder ◽

Negative Feedback ◽

Ryanodine Receptors ◽

Bk Channels ◽

Sk Channels ◽

Bladder Smooth Muscle ◽

Contraction Amplitude ◽

Contraction Frequency ◽

Urinary Bladder Smooth Muscle

This study examines the roles of voltage-dependent Ca2+ channels (VDCC), ryanodine receptors (RyRs), large-conductance Ca2+-activated K+ (BK) channels, and small-conductance Ca2+-activated K+ (SK) channels in the regulation of phasic contractions of guinea pig urinary bladder smooth muscle (UBSM). Nisoldipine (100 nM), a dihydropyridine inhibitor of VDCC, abolished spontaneous UBSM contractions. Ryanodine (10 μM) increased contraction frequency and thereby integrated force and, in the presence of the SK blocker apamin, had a greater effect on integrated force than ryanodine alone. Blocking BK (iberiotoxin, 100 nM) or SK (apamin, 100 nM) channels increased contraction amplitude and duration but decreased frequency. The contractile response to iberiotoxin was more pronounced than to apamin. The increases in contraction amplitude and duration to apamin were substantially augmented with ryanodine pretreatment. These results indicate that BK and SK channels have prominent roles as negative feedback elements to limit UBSM contraction amplitude and duration. RyRs also appear to play a significant role as a negative feedback regulator of contraction frequency and duration, and this role is influenced by the activity of SK channels.

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The large conductance Ca 2+ ‐ and voltage‐activated K + channel is the primary K + channel that controls cell excitability and contractility in human urinary bladder smooth muscle

The FASEB Journal ◽

10.1096/fasebj.23.1_supplement.781.3 ◽

2009 ◽

Vol 23 (S1) ◽

Author(s):

Kiril L. Hristov ◽

Muyan Chen ◽

Whitney F. Kellett ◽

Eric S. Rovner ◽

Georgi V. Petkov

Keyword(s):

Smooth Muscle ◽

Urinary Bladder ◽

K Channel ◽

Bladder Smooth Muscle ◽

Cell Excitability ◽

Human Urinary Bladder ◽

Urinary Bladder Smooth Muscle

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Mineralocorticoids decrease the activity of the apical small-conductance K channel in the cortical collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.00063.2005 ◽

2005 ◽

Vol 289 (5) ◽

pp. F1065-F1071 ◽

Cited By ~ 7

Author(s):

Yuan Wei ◽

Elisa Babilonia ◽

Hyacinth Sterling ◽

Yan Jin ◽

Wen-Hui Wang

Keyword(s):

Protein Tyrosine Kinase ◽

Collecting Duct ◽

K Channel ◽

Sk Channels ◽

Cortical Collecting Duct ◽

Channel Activity ◽

Patch Clamp Technique ◽

Herbimycin A ◽

Sk Channel ◽

Small Conductance

We used the patch-clamp technique to examine the effect of DOCA treatment (2 mg/kg) on the apical small-conductance K (SK) channels, epithelial Na channels (ENaC), and the basolateral 18-pS K channels in the cortical collecting duct (CCD). Treatment of rats with DOCA for 6 days significantly decreased the plasma K from 3.8 to 3.1 meq and reduced the activity of the SK channel, defined as NPo, from 1.3 in the CCD of control rats to 0.6. In contrast, DOCA treatment significantly increased ENaC activity from 0.01 to 0.53 and the basolateral 18-pS K channel activity from 0.67 to 1.63. Moreover, Western blot analysis revealed that DOCA treatment significantly increased the expression of the nonreceptor type of protein tyrosine kinase (PTK), cSrc, and the tyrosine phosphorylation of ROMK in the renal cortex and outer medulla. The possibility that decreases in apical SK channel activity induced by DOCA treatment were the result of stimulation of PTK activity was further supported by experiments in which inhibition of PTK with herbimycin A significantly increased NPo from 0.6 to 2.1 in the CCD from rats receiving DOCA. Also, when rats were fed a high-K (10%) diet, DOCA treatment did not increase the expression of c-Src and decrease the activity of the SK channel in the CCD. We conclude that DOCA treatment decreased the apical SK channel activity in rats on a normal-K diet and that an increase in PTK expression may be responsible for decreased channel activity in the CCD from DOCA-treated rats.

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