Overactive Bladder and Incontinence in the Absence of the BK Large Conductance Ca2+-activated K+ Channel

Abstract

BK large conductance voltage- and calcium-activated potassium channels respond to elevations in intracellular calcium and membrane potential depolarization, braking excitability of smooth muscle. BK channels are thought to have a particularly prominent role in urinary bladder smooth muscle function and therefore are candidate targets for overactive bladder therapy. To address the role of the BK channel in urinary bladder function, the gene mSlo1 for the pore-forming subunit of the BK channel was deleted. Slo–/– mice were viable but exhibited moderate ataxia. Urinary bladder smooth muscle cells of Slo–/– mice lacked calcium- and voltage-activated BK currents, whereas local calcium transients (“calcium sparks”) and voltage-dependent potassium currents were unaffected. In the absence of BK channels, urinary bladder spontaneous and nerve-evoked contractions were greatly enhanced. Consistent with increased urinary bladder contractility caused by the absence of BK currents, Slo–/– mice demonstrate a marked elevation in urination frequency. These results reveal a central role for BK channels in urinary bladder function and indicate that BK channel dysfunction leads to overactive bladder and urinary incontinence. BK large conductance voltage- and calcium-activated potassium channels respond to elevations in intracellular calcium and membrane potential depolarization, braking excitability of smooth muscle. BK channels are thought to have a particularly prominent role in urinary bladder smooth muscle function and therefore are candidate targets for overactive bladder therapy. To address the role of the BK channel in urinary bladder function, the gene mSlo1 for the pore-forming subunit of the BK channel was deleted. Slo–/– mice were viable but exhibited moderate ataxia. Urinary bladder smooth muscle cells of Slo–/– mice lacked calcium- and voltage-activated BK currents, whereas local calcium transients (“calcium sparks”) and voltage-dependent potassium currents were unaffected. In the absence of BK channels, urinary bladder spontaneous and nerve-evoked contractions were greatly enhanced. Consistent with increased urinary bladder contractility caused by the absence of BK currents, Slo–/– mice demonstrate a marked elevation in urination frequency. These results reveal a central role for BK channels in urinary bladder function and indicate that BK channel dysfunction leads to overactive bladder and urinary incontinence. Overactive urinary bladder and urinary incontinence is a significant health issue occurring in about 51 million (∼17%) of the United States population (1Hu T.W. Wagner T.H. Bentkover J.D. Leblanc K. Zhou S.Z. Hunt T. Urology. 2004; 63: 461-465Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), frequently occurring as a secondary consequence of conditions such as diabetes mellitus, stroke, and spinal cord injury. Urge incontinence is caused by overactivity of the urinary bladder smooth muscle (UBSM), 1The abbreviations and trivial terms used are: UBSM, urinary bladder smooth muscle; BK, large conductance calcium-activated K+ channel; IBTX, iberiotoxin; Slo–/–, mSlo1 homozygous null.1The abbreviations and trivial terms used are: UBSM, urinary bladder smooth muscle; BK, large conductance calcium-activated K+ channel; IBTX, iberiotoxin; Slo–/–, mSlo1 homozygous null. often a result of partial urethral outlet obstruction that can occur during prostate hypertrophy. Currently there is a lack of effective therapeutic agents to control urinary bladder function. Antimuscarinic agents, which impair UBSM contraction, are used to treat urinary incontinence but have limited effectiveness and undesirable side effects. More recently, potassium channel opening drugs have been explored as therapeutic agents for urinary incontinence (2Li J.H. Yasay G.D. Zografos P. Kau S.T. Ohnmacht C.J. Russell K. Empfield J.R. Brown F.J. Trainor D.A. Bonev A.D. Heppner T.J. Nelson M.T. Pharmacology. 1995; 51: 33-42Crossref PubMed Scopus (29) Google Scholar, 3Butera J.A. Antane S.A. Hirth B. Lennox J.R. Sheldon J.H. Norton N.W. Warga D. Argentieri T.M. Bioorg. Med. Chem. Lett. 2001; 11: 2093-2097Crossref PubMed Scopus (69) Google Scholar, 4Brune M.E. Fey T.A. Brioni J.D. Sullivan J.P. Williams M. Carroll W.A. Coghlan M.J. Gopalakrishnan M. J. Pharmacol. Exp. Ther. 2002; 303: 387-394Crossref PubMed Scopus (32) Google Scholar, 5Hewawasam P. Erway M. Thalody G. Weiner H. Boissard C.G. Gribkoff V.K. Meanwell N.A. Lodge N. Starrett Jr., J.E. Bioorg. Med. Chem. Lett. 2002; 12: 1117-1120Crossref PubMed Scopus (30) Google Scholar, 6Turner S.C. Carroll W.A. White T.K. Gopalakrishnan M. Coghlan M.J. Shieh C.C. Zhang X.F. Parihar A.S. Buckner S.A. Milicic I. Sullivan J.P. Bioorg. Med. Chem. Lett. 2003; 13: 2003-2007Crossref PubMed Scopus (24) Google Scholar). BK potassium channels regulate membrane potential and repolarization of UBSM action potentials (7Heppner T.J. Bonev A.D. Nelson M.T. Am. J. Physiol. 1997; 273: C110-C117Crossref PubMed Google Scholar, 8Hashitani H. Brading A.F. Br. J. Pharmacol. 2003; 140: 159-169Crossref PubMed Scopus (109) Google Scholar). UBSM BK channels are activated by membrane potential depolarization and calcium influx through voltage-dependent calcium channels that occur during the action potential (9Herrera G.M. Nelson M.T. J. Physiol. 2002; 541: 483-492Crossref PubMed Scopus (105) Google Scholar). UBSM BK channels are also activated by local intracellular calcium release through ryanodine receptors (calcium sparks) (10Herrera G.M. Heppner T.J. Nelson M.T. Am. J. Physiol. 2001; 280: C481-C490Crossref PubMed Google Scholar). Specific inhibition of BK channels by iberiotoxin (IBTX) causes a pronounced elevation in bladder contractility (8Hashitani H. Brading A.F. Br. J. Pharmacol. 2003; 140: 159-169Crossref PubMed Scopus (109) Google Scholar, 11Herrera G.M. Heppner T.J. Nelson M.T. Am. J. Physiol. 2000; 279: R60-R68Crossref PubMed Google Scholar, 12Petkov G.V. Bonev A.D. Heppner T.J. Brenner R. Aldrich R.W. Nelson M.T. J. Physiol. 2001; 537: 443-452Crossref PubMed Scopus (137) Google Scholar). Deletion of the smooth muscle-specific, modulatory β1 subunit decreases the apparent voltage- and calcium-sensitivity of UBSM BK channels, leading to an enhancement of phasic contractility (12Petkov G.V. Bonev A.D. Heppner T.J. Brenner R. Aldrich R.W. Nelson M.T. J. Physiol. 2001; 537: 443-452Crossref PubMed Scopus (137) Google Scholar). These studies point to a pivotal role of the BK channel in urinary bladder function. Generation of Slo–/–Mice—mSlo1 genomic clones were isolated from a 129/SvJ BAC library (Incyte Genomics) using an ∼1.6-kb EcoRV/XhoI cDNA probe fragment. A 7.8-kb SalI/BamHI genomic fragment containing exon 1 was cloned into pPNT (13Tybulewicz V.L. Crawford C.E. Jackson P.K. Bronson R.T. Mulligan R.C. Cell. 1991; 65: 1153-1163Abstract Full Text PDF PubMed Scopus (1156) Google Scholar, 14Joyner A.L. Gene Targeting: A Practical Approach. Oxford University Press, Oxford1993: 1-61Google Scholar). Synthetic double-stranded oligos containing loxP recognition sequences for Cre recombinase were cloned 5′ (XhoI site, 5′-TCCCTCGAATAACTTCGTATAGCATACATTATACGAAGTTATTCGAGCCC-3′) and 3′ (NcoI site, 5′-GGATCCATAACTTCGTATAGCATACATTATACGAAGTTATCCATGG-3′) of exon 1. Enhanced green fluorescent protein containing β-globin basal promoter elements and a bovine growth hormone polyadenylation signal (a gift from Dr. Jane Johnson) was subcloned into the mSlo1 intronic sequence at the NcoI site. For removal of the neo cassette, oligos containing FRT recognition sites for Flp recombinase were subcloned into the XhoI and XbaI sites flanking PGKneo (5′-CTCGATGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCTCGAG-3′ and 5′-TCTAGGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAGATCC-3′). An ∼1.2-kb BamHI/KpnI mSlo1 genomic fragment was subcloned 3′ of the neo cassette into XbaI/KpnI of pPNT. The targeting construct was linearized for transfection with NotI and electroporated into R1 embryonic stem cells. After ganciclovir and G418 selections, six correct integrants were identified by PCR. To verify intact integration of the entire targeting construct, these clones were analyzed by Southern blot using external 5′ and 3′ probes on XhoI and BamHI/MfeI-digested genomic DNA, respectively. Two correctly integrated clones were injected into C57Bl/6 blastocyst stage embryos, producing five germ line-transmitting founders. To generate a whole animal Slo–/– mutant, founder males were crossed to FVB-TgN(EIIa-Cre)C5379Lmgd females (Jackson Laboratories), a line that ubiquitously expresses Cre from the adenoviral EIIa promoter. All F1 progeny had stable, germ line-transmissible deletions of exon 1, as confirmed by Southern blot analysis (Fig. 1B). Heterozygotes were backcrossed >6 generations to FVB/NJ. Inter-crosses to obtain homozygous Slo–/– were genotyped by PCR on tail DNA. PCR Genotyping of Slo–/–Mice—Tail snips were digested overnight at 55 °C in 750 μl of SNET (20 mm Tris, pH 8, 1 mm EDTA, 1% SDS, 0.4 m NaCl) plus 15 μl of 20 mg/ml proteinase K and extracted with an equal volume 1:1 phenol/chloroform. DNA was ethanol-precipitated and 500 ng of DNA (or 2 μl of supernatant) was used in PCR reactions (NEB Taq, supplier's reaction condition plus 2% Me2SO; amplification conditions: 94 °C, 2 min; (94 °C, 30 s; 55–50 °C, 30 s; 72 °C, 2 min) × 5 cycles; (94 °C, 30 s; 50 °C, 30 s; 72 °C, 2 min) × 30 cycles; 72 °C, 5′; 4 °C, hold). Neo 5′ (5′-ATA GCC TGA AGA ACG AGA TCA GC-3′) and RA 14025 3′ (5′-CCT CAA GAA GGG GAC TCT AAA C-3′) amplify the Slo–/– allele product of 800 bp. Exon 1 5′-3 (5′-TTC ATC ATC TTG CTC TGG CGG ACG-3′) and WT 3′-2 (5′-CCA TAG TCA CCA ATA GCC C-3′) amplify the wild-type product of 332 bp. Isolation of UBSM and Western Blotting—All procedures involving mice were in accordance with Institutional Animal Care and Use Committee policies at the University of Vermont and Stanford University. Slo+/+ and Slo–/– mice 1.5–3 months of age were sacrificed by intraperitoneal injection of a lethal dose of sodium pentobarbital (150 mg/kg) or CO2 euthanasia. For Western blots, urinary bladders were solubilized in lysis buffer (137 mm NaCl, 1% Triton X-100, 0.5% deoxycholate, 40 mm HEPES, pH 7.4, 1 mm EDTA, pH 7.4, 2 μg/ml aprotinin, 1 μg/ml leupeptin, 2 μg/ml antipain, 10 μg/ml benzamidine, and 0.5 mm phenylmethylsulfonyl fluoride). The insoluble fraction was separated by centrifugation (14,000 × g for 5 min). 5 μg of soluble supernatant protein (assayed by Bio-Rad DC protein assay reagent) underwent SDS-PAGE on a 7.5% acrylamide gel prior to being transferred to a nitrocellulose membrane. Membranes were blocked in 4% dry nonfat milk, 2% normal goat serum, 10 mm Tris (pH 8), 0.15 m NaCl, and 0.1% Tween 20 for 1 h. They were then labeled with primary antibodies in blocking solution overnight, 1:5000 each of rabbit polyclonal α-Slo (Alamone Labs, Jerusalem, Israel) and mouse monoclonal DM 1A α-tubulin (Sigma). Membranes were labeled with 1:500 SuperSignal West Dura horseradish peroxidase-conjugated goat α-rabbit and α-mouse secondary antibodies (Pierce), and proteins were visualized by SuperSignal chemiluminescence detection (Pierce). Electrophysiology—UBSM cells from Slo+/+ and Slo–/– mice were isolated enzymatically for perforated whole-cell patch clamp recordings at 22 °C as previously described (15Thorneloe K.S. Nelson M.T. J. Physiol. 2003; 549: 65-74Crossref PubMed Scopus (65) Google Scholar, 16Horn R. Marty A. J. Gen. Physiol. 1988; 92: 145-159Crossref PubMed Scopus (1450) Google Scholar). The bath and pipette solutions contained the following (mm): 134 NaCl, 6 KCl, MgCl2, 2 CaCl2, 10 glucose, and 10 Hepes, pH 7.4 (NaOH), and 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 0.05 EGTA, 200 μgml–1 amphotericin B, and 10 Hepes, pH 7.2 (KOH), respectively. Iberiotoxin (Sigma) was used at a final concentration of 100 or 300 nm. Ca2+Spark Measurements—Isolated UBSM cells were loaded on glass coverslips with the Ca2+-sensitive fluorophore, fluo 4-AM (Molecular Probes), in cell isolation solution (16Horn R. Marty A. J. Gen. Physiol. 1988; 92: 145-159Crossref PubMed Scopus (1450) Google Scholar) with 10 μm fluo 4-AM, 0.04% pluronic acid, and 2 mm CaCl2 for 20 min. After loading, UBSM cells were washed with electrophysiology bath solution, and Ca2+ imaging was conducted on a laser-scanning confocal microscope (OZ; Noran Instruments) at room temperature, acquiring images at 120 or 30 Hz with scan durations of 25 s. Ca2+ sparks were defined as local increases in fluorescence of 1.3 F/Fo (F, instantaneous; Fo, baseline fluorescence) that persisted for at least two images and were analyzed with custom software written by Dr. Adrian Bonev using IDL Research Systems (17Perez G.J. Bonev A.D. Patlak J.B. Nelson M.T. J. Gen. Physiol. 1999; 113: 229-238Crossref PubMed Scopus (234) Google Scholar, 18Jaggar J.H. Nelson M.T. Am. J. Physiol. 2000; 279: C1528-C1539Crossref PubMed Google Scholar, 19Wellman G.C. Santana L.F. Bonev A.D. Nelson M.T. Am. J. Physiol. 2001; 281: C1029-C1037Crossref PubMed Google Scholar). Fo was obtained by averaging 10 images that displayed no Ca2+ sparks. For quantitation of Ca2+ sparks, a box (10 × 10 pixels, 2.2 × 2.2 μm) was placed over the Ca2+ spark site, and F/Fo traces were generated. Contractility Studies—Contractility experiments were performed at 37 °C as previously described (20Herrera G.M. Pozo M.J. Zvara P. Petkov G.V. Bond C.T. Adelman J.P. Nelson M.T. J. Physiol. 2003; 551: 893-903Crossref PubMed Scopus (107) Google Scholar) using a MyoMed myograph system (MED Associates Inc.). Electrical field stimulation was at 20 Hz (20 V amplitude, alternating polarity between pulses, 0.2-ms stimulation width) delivered once a minute for 2 s. Electrical field stimulation evoked a robust contraction of UBSM strips that was completely blocked by 1 μm tetrodotoxin (n was in and to the solution at a concentration of 100 nm. phasic was analyzed using Inc.). mice months of age were placed in for 1 with the by and were were PubMed Scopus Google Scholar, G. G. M.J. 2002; PubMed Scopus Google Scholar). are as were with the and were Slo–/– were Generation of the that BK channel dysfunction leads to overactive bladder and urinary a of the pore-forming by the mSlo1 by in embryonic stem cells using A.L. Gene Targeting: A Practical Approach. Oxford University Press, Oxford1993: 1-61Google Scholar). A allele of the mSlo1 gene was by flanking exon 1 with loxP which of exon 1 by of Cre recombinase (Fig. Exon 1 the as as the of the channel for of channels M. P. A. PubMed Scopus Google Scholar). of Cre recombinase exon 1, a stable, germ line-transmissible allele (Fig. mSlo1 (n and protein (Fig. are in UBSM from Slo–/– the allele is a null. of the of Slo–/– mice using and mSlo1 is P. 2002; PubMed Scopus Google Scholar) and BK channels are to in Slo–/– mice normal (n and (n with of and Scholar). Slo–/– mice are in normal (Fig. and Slo–/–, are Slo+/+ at 2 7.2 Slo–/–, by 5 of age is to 0.5 0.5 Slo–/–, an of Slo–/– mice at an age of 2.2 months of causes (n The mice have normal Slo–/– mice are to the of is 1 of 20 Slo–/– males for to wild-type was to a of normal 1 the role of BK channels in smooth muscle during K. G.J. N. M. Am. J. Physiol. PubMed Google Scholar, M. N. R. B. Lett. 1999; PubMed Scopus Google Scholar), Slo–/– females can to and and for from females to 0.4 Slo–/– 0.5 The by Slo–/– mice is moderate ataxia. analysis that Slo–/– mice have a (Fig. The of a was analyzed for each mouse by and through a on with of and Scholar). Slo+/+ mice had an of (n 5 whereas Slo–/– mice displayed an with a of (n In a of Slo–/– mice an at with (Fig. is (Fig. and Slo–/– mice also in the assay with of and Scholar), a as as for which mice an and the is (Fig. between in central basal or or but of these is by or in Slo–/– mice (n The of the is being with Cre and the of also spontaneous (n is of a on Slo–/– mice reveal basal or (n in cells isolated from Slo+/+ mice a pronounced BK channel as the voltage-activated by IBTX, a of BK channels (Fig. BK channel currents are in UBSM cells from Slo–/– mice (Fig. the of UBSM cells isolated from Slo+/+ and Slo–/– The whole-cell was cell 40 4 Slo–/– K+ currents using a K+ were at (Fig. Slo–/– UBSM K+ was blocked by with 10 mm (Fig. and These results are with that for the mouse UBSM K+ (15Thorneloe K.S. Nelson M.T. J. Physiol. 2003; 549: 65-74Crossref PubMed Scopus (65) Google Scholar). in channels are activated by elevations in intracellular calcium and in by local and calcium release (calcium sparks) through ryanodine receptors in the membrane (9Herrera G.M. Nelson M.T. J. Physiol. 2002; 541: 483-492Crossref PubMed Scopus (105) Google Scholar, G.M. Heppner T.J. Nelson M.T. Am. J. Physiol. 2001; 280: C481-C490Crossref PubMed Google Scholar, G.J. Bonev A.D. Patlak J.B. Nelson M.T. J. Gen. Physiol. 1999; 113: 229-238Crossref PubMed Scopus (234) Google Scholar, M.T. H. M. Santana L.F. Bonev A.D. 1995; PubMed Scopus Google Scholar, N. K. H. K. M. J. Physiol. PubMed Scopus Google Scholar, K. M. Am. J. Physiol. PubMed Google Scholar, H. N. K. M. J. Physiol. 2001; PubMed Scopus Google Scholar). UBSM cells isolated from Slo+/+ mice BK currents of by calcium sparks. These are by with (Fig. A and Consistent with the lack of voltage-activated BK currents in UBSM cells of Slo–/– mice (Fig. BK currents were in Slo–/– mice (Fig. A and These indicate that the mSlo1 gene the BK channel activated by and calcium in A lack of BK currents in Slo–/– mice ryanodine (calcium calcium sparks were in Slo–/– UBSM cells from Slo+/+ and Slo–/– mice were loaded with the Ca2+-sensitive and in intracellular Ca2+ were using a laser-scanning confocal local increases in Ca2+ in UBSM cells from Slo+/+ and Slo–/– mice (Fig. and with and with calcium sparks described previously (10Herrera G.M. Heppner T.J. Nelson M.T. Am. J. Physiol. 2001; 280: C481-C490Crossref PubMed Google Scholar, G.J. Bonev A.D. Patlak J.B. Nelson M.T. J. Gen. Physiol. 1999; 113: 229-238Crossref PubMed Scopus (234) Google Scholar, N. K. H. K. M. J. Physiol. PubMed Scopus Google Scholar, K. M. Am. J. Physiol. PubMed Google Scholar, H. N. K. M. J. Physiol. 2001; PubMed Scopus Google Scholar). Ca2+ spark and were between Slo+/+ and Slo–/– UBSM cells (Fig. and and there were no in the of spark cell Slo–/– Ca2+ spark Slo–/– or in the of Ca2+ spark Slo+/+ Slo–/– Enhanced UBSM Contractility to Urinary the role of BK channels in urinary bladder contractility isolated UBSM UBSM strips spontaneous phasic contractions that are for the of urinary bladder to overactive bladder G.J. M. T. J. K. K. M. A. Am. J. Physiol. 2001; 281: Google Scholar). contraction amplitude, and were increased in Slo–/– over Slo+/+ mice (Fig. phasic × was increased in Slo–/– with Slo+/+ is caused by stimulation of in the bladder To the role of BK channels in urinary bladder contraction, field stimulation at a to release in UBSM the that during BK channels with in Slo+/+ UBSM strips in a enhancement of an that was in Slo–/– strips (Fig. and contractions of UBSM strips from Slo–/– mice were the by Slo+/+ strips (Fig. and the during membrane potential depolarization by to mm was between Slo+/+ and Slo–/– strips the to the in each UBSM a in contractions in Slo–/– UBSM (Fig. in to increased phasic contractility in Slo–/– mice (Fig. contractions are also BK channels are (Fig. The of BK channels in UBSM and the robust in UBSM contractility by Slo–/– mice that BK channels for normal bladder function in To for urinary bladder overactivity in the absence of BK channels, the urination of Slo+/+ and Slo–/– mice were Slo+/+ mice an of in an from Slo–/– mice (Fig. Slo–/– mice Slo+/+ Slo–/– mice exhibited (n which was in Slo+/+ (n significant in Slo–/– or as Slo–/– were that for the increased urination in Slo–/– that the overactivity in Slo–/– bladders is a result of increased by the but is a in the urinary bladder caused by the absence of BK In have that the absence of BK currents basal and UBSM contractility (Fig. leading to bladder overactivity and urinary incontinence (Fig. BK channels in to depolarization and increases in intracellular Ca2+ during UBSM action excitability and as in Slo–/– excitability leading to muscle a primary of urinary incontinence. the of bladder is and a of that BK channels a therapeutic for and causes of bladder The increased of spontaneous contractions in Slo–/– mice that by BK channels in with causes for incontinence bladder the increased of contractions in to a in UBSM of Slo–/– mice that with caused incontinence also from BK channel Consistent with of cDNA into urinary bladder bladder overactivity caused by partial urethral outlet obstruction G.J. M. T. J. K. K. M. A. Am. J. Physiol. 2001; 281: Google Scholar). the of BK channel J.A. Antane S.A. Hirth B. Lennox J.R. Sheldon J.H. Norton N.W. Warga D. Argentieri T.M. Bioorg. Med. Chem. Lett. 2001; 11: 2093-2097Crossref PubMed Scopus (69) Google Scholar, 5Hewawasam P. Erway M. Thalody G. Weiner H. Boissard C.G. Gribkoff V.K. Meanwell N.A. Lodge N. Starrett Jr., J.E. Bioorg. Med. Chem. Lett. 2002; 12: 1117-1120Crossref PubMed Scopus (30) Google Scholar, 6Turner S.C. Carroll W.A. White T.K. Gopalakrishnan M. Coghlan M.J. Shieh C.C. Zhang X.F. Parihar A.S. Buckner S.A. Milicic I. Sullivan J.P. Bioorg. Med. Chem. Lett. 2003; 13: 2003-2007Crossref PubMed Scopus (24) Google Scholar) an effective for the of bladder overactivity in the undesirable side of these results that BK channel dysfunction can to overactive bladder and urinary incontinence. for with mouse for and and Dr. for