SK3-1C, a Dominant-negative Suppressor of SKCa and IKCa Channels

Kolski-Andreaco, Aaron; Tomita, Hiroaki; Shakkottai, Vikram G.; Gutman, George A.; Cahalan, Michael D.; Gargus, J. Jay; Chandy, K. George

doi:10.1074/jbc.m311725200

Cited by 33 publications

(37 citation statements)

References 64 publications

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“…As a result of the multimeric assembly nature of SK channels, this leads to the possibility of heteromultimeric assembly in which more than one SK family member (SK1-3) may assemble into a given channel complex. This has been demonstrated in heterologous expression systems (1,17,20,26) and directly demonstrated in native tissue using immunoprecipitation (33). In addition, the transgenic expression of a dominant negative SK subunit supports the heteromultimeric assembly of SK channels in vivo (38).…”

Section: Discussionmentioning

confidence: 75%

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

Thorneloe¹,

Knorn²,

Doetsch³

et al. 2008

American Journal of Physiology-Regulatory, Integrative and Comp

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-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...

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Section: Discussionmentioning

confidence: 75%

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

Thorneloe¹,

Knorn²,

Doetsch³

et al. 2008

American Journal of Physiology-Regulatory, Integrative and Comp

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“…As illustrated in Figure 2, expression of SK3-1B effectively inhibited currents carried through K Ca 2.1-K Ca 2.3 as well as K Ca 3.1 channels ( p Ͻ 0.01 for each of the currents) but had little effect on currents carried through K Ca 1.1. Previous work has shown that SK3-1B expression is without effect on currents carried through voltagedependent potassium channels, and its dominant-negative activity is not attributable to dysregulated calmodulin homeostasis (Tomita et al, 2003;Kolski-Andreaco et al, 2004;. Thus, SK3-1B appears to be a potent tool to assess the involvement of SK Ca -IK Ca subunits in the formation of the channels responsible for I sAHP .…”

Section: Resultsmentioning

confidence: 97%

“…Whole-cell recordings for SK Ca , IK Ca , and BK Ca currents were performed using an internal solution containing 1 M free Ca 2ϩ (in mM: 145 K-aspartate, 8.5 CaCl 2 , 10 EGTA, 10 HEPES, and 2 MgCl 2 , pH 7.4), as described previously (Kolski-Andreaco et al, 2004). The external recording solution contained the following (in mM): 155 Na ϩ aspartate, 4.5 KCl, 2 CaCl 2 , 1 MgCl 2 , and 10 HEPES, pH 7.2, 280 -300 mOsm.…”

Section: Methodsmentioning

confidence: 99%

SK_CaChannels Mediate the Medium But Not the Slow Calcium-Activated Afterhyperpolarization in Cortical Neurons

Villalobos¹,

Shakkottai²,

Chandy³

et al. 2004

J. Neurosci.

114

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Many neurons, including pyramidal cells of the cortex, express a slow afterhyperpolarization (sAHP) that regulates their firing. Although initial findings suggested that the current underlying the sAHP could be carried through SK Ca channels, recent work has uncovered anomalies that are not congruent with this idea. Here, we used overexpression and dominant-negative strategies to assess the involvement of SK Ca channels in mediating the current underlying the sAHP in pyramidal cells of the cerebral cortex.Pyramidal cells of layer V exhibit robust AHP currents composed of two kinetically and pharmacologically distinguishable currents known as the medium AHP current (I mAHP ) and the slow AHP current (I sAHP ). I mAHP is blocked by the SK Ca channel blockers apamin and bicuculline, whereas I sAHP is resistant to these agents but is inhibited by activation of muscarinic receptors. To test for a role for SK Ca channels, we overexpressed K Ca 2.1 (SK1) and K Ca 2.2 (SK2), the predominant SK Ca subunits expressed in the cortex, in pyramidal cells of cultured brain slices. Overexpression of K Ca 2.1 and K Ca 2.2 resulted in a fourfold to fivefold increase in the amplitude of I mAHP but had no detectable effect on I sAHP . As an additional test, we examined I sAHP in a transgenic mouse expressing a truncated SK Ca subunit (SK3-1B) capable of acting as a dominant negative for the entire family of SK Ca -IK Ca channels. Expression of SK3-1B profoundly inhibited I mAHP but again had no discernable effect on I sAHP . These results are inconsistent with the proposal that SK Ca channels mediate I sAHP in pyramidal cells and indicate that a different potassium channel mediates this current.

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“…The reason why efficiency is different remains to be elucidated. Previous reports described several SK3 mRNA variants with distinct sequences for exon 1 that encode SK3 proteins with distinct NH 2 termini (25,26). It is not excluded that high Dexon3 siRNA efficiency is due to the full targeting and silencing of SK3 isoforms, in contrast to Dexon1 siRNA.…”

Section: Whereas Sk3 Gene Transcript Destruction Decreased Migration mentioning

confidence: 99%

Identification of SK3 channel as a new mediator of breast cancer cell migration

Potier

Joulin²,

Roger³

et al. 2006

Molecular Cancer Therapeutics

118

121

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Potassium channels have been involved in epithelial tumorigenesis but the role of small-conductance Ca 2+ -activated K + channels is unknown. We report here that small-conductance Ca 2+ -activated K + channels are expressed in a highly metastasizing mammary cancer cell line, MDA-MB-435s. Patch-clamp recordings showed typical small-conductance Ca 2+ -activated K + channelmediated currents sensitive to apamin, 4-aminopyridine, and tetraethylammonium. Moreover, the cells displayed a high intracellular calcium concentration, which was decreased after 24 hours of apamin treatment. By regulating membrane potential and intracellular calcium concentration, these channels were involved in MDA-MB435s cell migration, but not in proliferation. Only SK3 protein expression was observed in these cells in contrast to SK2, which was expressed both in cancer and noncancer cell lines. Whereas small interfering RNA directed against SK3 almost totally abolished MDA-MB435s cell migration, transient expression of SK3 increased migration of the SK3-deficient cell lines, MCF-7 and 184A1. SK3 channel was solely expressed in tumor breast biopsies and not in nontumor breast tissues. Thus, SK3 protein channel seems to be a new mediator of breast cancer cell migration and represents a potential target for a new class of anticancer agents.

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SK3-1C, a Dominant-negative Suppressor of SKCa and IKCa Channels

Cited by 33 publications

References 64 publications

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

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

SK_CaChannels Mediate the Medium But Not the Slow Calcium-Activated Afterhyperpolarization in Cortical Neurons

Identification of SK3 channel as a new mediator of breast cancer cell migration

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SK3-1C, a Dominant-negative Suppressor of SKCa and IKCa Channels

Cited by 33 publications

References 64 publications

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

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

SKCaChannels Mediate the Medium But Not the Slow Calcium-Activated Afterhyperpolarization in Cortical Neurons

Identification of SK3 channel as a new mediator of breast cancer cell migration

Contact Info

Product

Resources

About

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

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

SK_CaChannels Mediate the Medium But Not the Slow Calcium-Activated Afterhyperpolarization in Cortical Neurons