Generation of Kcnma1fl-tdTomato, a conditional deletion of the BK channel α subunit in mouse

BK large conductance calcium-activated K+ channels (KCa1.1) are expressed widely across many tissues, contributing to systemic regulation of cardiovascular, neurological, and other specialized physiological functions. The pore-forming α subunit is encoded by the Kcnma1 gene, originally named mSlo1 in mouse and slowpoke in Drosophila. Global deletion in mouse (Kcnma1−/−) produces a plethora of defects in neuron and muscle excitability, as well as other phenotypes related to channel function in nonexcitable cells. While homozygous null mice are viable, the ubiquitous loss of BK function has complicated the interpretation of phenotypes involving the interaction of multiple cell types which independently express BK channels. Here, we report the generation of a targeted allele for conditional inactivation of Kcnma1 using the Cre-loxP system (Kcnma1fl-tdTomato). Cre-mediated recombination generates a null allele, and BK currents were not detectable in neurons and muscle cells from Nestin-Cre; Kcnma1fl/fl and SM22α-Cre; Kcnma1fl/fl mice, respectively. tdTomato expression was detected in Cre-expressing tissues, but not in Cre-negative controls. These data demonstrate the utility of Kcnma1fl-tdTomato for conditional deletion of the BK channel, facilitating the understanding of tissue-specific contributions to physiological function in vivo.

Global deletions of Kcnma1 (Kcnma1 À/À ) have been indispensable for understanding how BK currents regulate cellular and integrated physiology in mammals, revealing the essential function of BK channels in neurons, muscle, and nonexcitable cells in vivo. Kcnma1 À/À mice have alterations in circadian rhythm, heart rate, blood pressure, urination, locomotion, reproductive function, neurovascular coupling, hearing, airway constriction, insulin secretion, and neurological learning behaviors (Meredith et al. 2004Ruttiger et al. 2004;Sausbier et al. 2004Sausbier et al. , 2005Sausbier et al. , 2007Werner et al. 2005;Filosa et al. 2006;Pyott et al. 2007;Dufer et al. 2011;Typlt et al. 2013;Lai et al. 2014). These mice were further used to demonstrate that BK channels are the targets of a fungal neurotoxin that causes Ryegrass Staggers (Imlach et al. 2008) and can be localized to intracellular organelles and to complexes containing Ca 2+ channels (Indriati et al. 2013;Singh et al. 2013;Li et al. 2014;Cao et al. 2015). However, the ubiquitous loss of BK function in several systems has made interpretation of phenotypes challenging. For example, the ataxia in Kcnma1 À/À mice initially confounded analysis of circadian rhythms in locomotor activity ) and the contribution of BK channels to vascular hypertension was complicated by hyperaldosteronism (Sausbier et al. 2005). Furthermore, aspects of cardiac, bladder, and renal function are compensated in Kcnma1 À/À mice (Rieg et al. 2007;Sprossmann et al. 2009;Lai et al. 2014). Thus, to provide a higher resolution picture for the distinct contributions of BK channels in particular tissues to changes in physiology, we generated a targeted floxed allele, Kcnma1 fl -tdTomato. BK currents and tdTomato fluorescence were evaluated in neurons and smooth muscle, two tissue types exhibiting the highest endogenous expression levels, using Nestin-Cre and SM22a-Cre drivers, respectively (Tronche et al. 1999;Lepore et al. 2005).

Materials and Methods
Generation of Kcnma1 fl -tdTomato mice A targeting vector was created that contained 10.95 kb of Kcnma1 genomic sequence flanking exon 2 (B6 BAC clone RP23: 64P21) subcloned into pSP72, containing an ampicillin selection cassette (Ingenious Targeting Laboratory, Ronkonkoma, NY). A Lox71 site was subcloned 3 0 of the 6.02-kb long homology arm (LA), 312-bp upstream of exon 2. A mini-gene was generated consisting of a Lox66 site, 255-bp intron sequence, 33 bp of exon 2, the 2A-tdTomato sequence, followed by a bovine growth hormone polyadenylation sequence (BGHpA). The final 2A-tdTomato cassette was inserted into the targeting vector using MluI and SalI sites. The short homology arm (SA) extends 3.77 kb 3 0 to the mini gene. A pGK-gb2 FRT-flanked neomycin resistance cassette was subcloned 327-bp downstream of exon 2, 5 0 to a 2A-tdTomato cassette in reverse orientation. The targeting vector was confirmed by restriction analysis and sequencing after each modification. The total targeting vector size was 17.13 kb, and the construct was linearized with NotI for electroporation into BA1 (C57BL/ 6 9 129/SvEv) embryonic stem cells (Ingenious Targeting Laboratory).

Mice
All procedures involving mice were conducted in accordance with The University of Maryland School of Medicine animal care and use guidelines. Experimental mice were F2 Cre-positive (SM22a-Cre or Nes-Cre) or Crenegative homozygous Kcnmal fl/fl littermates. To harvest tissues for protein isolation and electrophysiology, mice were euthanized by inhalation of saturating isofluorane vapors, followed by rapid decapitation.

Western blot analysis
Protein was isolated from mouse (3-4 months) urinary bladders and subjected to western blotting as described previously (White et al. 2014). BK bands were detected with 1:1000 rabbit polyclonal a-BK (APC-021, Alamone Labs, Jerusalem, Israel) and visualized with the Super-Signal West Dura Kit 1:500 horseradish peroxidaseconjugated goat anti-rabbit secondary antibody (Pierce). a-Tubulin was detected by 1:10,000 DM1A mouse monoclonal a-tubulin (Sigma). BK band intensity was normalized to DM1A.

tdTomato imaging
All tissues were freshly dissected and unfixed. Bladder tissue strip images were acquired with a Zeiss LSM710 confocal microscope using a 209 objective, 561 nm excitation, and 580-703 nm emission filter. Equivalent acquisition settings were used for each genotype (Gain = 939). For analysis of fluorescence intensity, the average pixel intensities were calculated from 12 equivalently sized boxes per mouse (n = 3 mice). SCN images were similarly acquired from thick section coronal brain slices, prepared as described previously (Montgomery et al. 2013), using equivalent acquisition settings between genotypes (Gain = 939). For analysis, average pixel intensities were calculated from 4 to 8 equivalently sized boxes per mouse (n = 3 mice).

Results
Generation of the conditional Kcnma1 fl -tdTomato allele  (2002). The human gene structure is conserved on chromosome 10 (NC_000010.11; Gene ID: 3778). Two prior knockout transgenic lines were generated by targeting exon 1, containing the S0 domain (Meredith et al. 2004), or exon 9, containing the pore domain . In this study, the second exon was targeted for Cre/ loxP-mediated recombination. A targeting vector was constructed using an "inversion" strategy to generate the conditional inactivation (Meredith 2015). This event consists of Cre-mediated inversion of the second exon of Kcnma1, with simultaneous "knockin" of a tdTomato reporter (Shaner et al. 2004) that was placed in antiparal-lel (reverse) orientation in intron 2. The allele retains wild-type (WT) expression of Kcnma1 from the endogenous promotor and regulatory elements, and the reverse orientation of the 2A-tdTomato cassette will not be expressed, until Cre-mediated recombination inverts the cassette. The inversion is mediated by two mutant loxP sites, flanking exon 2 and the reverse orientation 2A-tdTomato cassette in intron 2 (Fig. 1B). The mutant loxP sequences cannot undergo repeated recombination after the initial Cre-mediated inversion event. In this way, the inactivated Kcnma1 allele is stably maintained in Creexpressing tissues (Meredith 2015). The inversion event renders exon 2 unable to be transcribed, while bringing the 2A-tdTomato cassette into the forward orientation and placing it under the control of the endogenous Kcnma1 promotor and regulatory elements. After transcription of the exon 1, the 2A peptide sequence mediates cotranslational cleavage via a "ribosome skipping" mechanism, allowing expression of tdTomato as a separate polypeptide (Fang et al. 2005) (Szymczak et al. 2004). tdTomato fluorescence will thus identify cells that undergo Cre-mediated Kcnma1 inactivation. Founder mice carrying the unrecombined allele were first mated to mice expressing FLPe recombinase under the control of the human ACTB promoter (Rodriguez et al. 2000) to remove the neomycin selection cassette (Fig. 1C). Homozygous Kcnma1 fl/fl progeny that harbored a deletion of the neocassette were grossly normal, with no  obvious health or viability differences compared to nontransgenic littermates (unpublished observations). Kcn-ma1 fl/fl mice were mated to either Nestin-Cre (Tronche et al. 1999) or SM22a-Cre mice (Lepore et al. 2005), and intercrossed to produce Cre-positive homozygous Kcn-ma1 fl/fl (Fig. 1C).
Smooth muscle-specific BK channel inactivation (SM22a-Cre; Kcnma1 fl/fl ) BK channels are highly expressed in smooth muscle, especially urinary bladder (Meredith et al. 2004). Therefore, we tested the functionality of the floxed allele in smooth muscle to determine whether BK protein was eliminated by Cre-mediated recombination. BK expression was analyzed by western blot in bladder tissue harvested from WT, SM22a-Cre-negative; Kcnma1 fl/fl , SM22a-Cre-positive; Kcnma1 fl/fl , and Kcnma1 À/À mice carrying a global deletion (Meredith et al. 2004). The cytoskeletal SM22a promoter drives expression in most smooth muscle cells, including bladder (Lepore et al. 2005). BK expression was comparable between WT and Cre-negative; Kcnma1 fl/fl UBSM tissue ( Fig. 2A). In contrast, negligible BK channel detectability was observed in Cre-positive; Kcnma1 fl/fl bladder tissue, similar to levels in the previously characterized Kcnma1 À/À functional null mice ( Fig. 2A, B). This residual BK detectability in Cre-positive; Kcnma1 fl/fl tissue could stem from less than 100% efficiency of Cre-mediated recombination with SM22a-Cre in UBSM cells, or incomplete removal of the nonsmooth muscle urothelial layer, which expresses BK channels. In aggregate, these data show that BK protein expression is essentially completely gone after Cre-mediated inversion of exon 2 and confirm that in the absence of Cre expression, BK protein levels are normal. Next to assess tdTomato expression, fluorescence was compared between SM22a-Cre-negative and -positive; Kcnma1 fl/fl bladder strips. In the absence of Cre, very little tdTomato fluorescence was observed, while Cre-positive animals exhibited strong fluorescence (Fig. 2C, D). These data indicate that tdTomato is both easy to distinguish from controls and also not significantly expressed unless Cre-mediated recombination occurs.
BK currents were assessed from smooth muscle cells of mesenteric artery, a cell type where the majority of the total K + current is due to BK channels (Fig. 3A). In Crenegative; Kcnma1 fl/fl cells, large outward currents were evoked by depolarizing voltage steps, and this current was sensitive to paxilline, a BK channel antagonist (Fig. 3B) (Knaus et al. 1994;Gribkoff et al. 1996). In contrast, Crepositive; Kcnma1 fl/fl cells did not have large evoked currents and were of similar magnitude to Cre-negative cells after paxilline block (Fig. 3C-F). These data confirm that BK currents are detectable from homozygous Kcnma1 fl/fl smooth muscle cells in the absence of Cre and are entirely removed when Cre is expressed.  Neuronal-specific BK channel inactivation (Nestin-Cre; Kcnma1 fl/fl ) We further confirmed the ability of Cre recombinase to recombine the Kcnma1 fl allele in neurons using a Nestin-Cre mouse line that expresses Cre throughout central and peripheral neurons (Tronche et al. 1999). We recorded macroscopic BK currents in whole-cell patch-clamp mode from neurons of the suprachiasmatic nucleus (Montgomery et al. 2013). In neurons, the BK current comprises a smaller component of the total K + current. Paxilline-sensitive BK currents were present in Cre-negative; Kcnma1 fl/fl SCN neurons (Fig. 4A, B), but not in Cre-positive neurons (Fig. 4C, D). tdTomato fluorescence was higher in Cre-positive neurons than Cre-negative controls (Fig. 4E), although some red fluorescence was detected in Cre-negative; Kcnma1 fl/fl slices and likely stems from autofluorescence (Schnell et al. 1999). These data corroborate the functionality of the Kcnma1 fl allele in a second tissue.

Discussion
The goal of this study was to demonstrate the null functionality of the Kcnma1 fl allele and the expression of tdTomato in the presence of Cre recombinase expression. Neither smooth muscle-(SM22a-Cre) nor neuron-specific (Nestin-Cre) BK channel deletions exhibit gross developmental, viability, or neurological (ataxia or tremor) phenotypes (unpublished observations). This is in contrast to the global BK channel deletion mice (Kcnma1 À/À ), which exhibit increased mortality, ataxia, and tremor (Meredith et al. 2004). The loss of BK currents and protein, and corresponding expression of tdTomato, was demonstrated in Cre-positive mice. BK protein and currents were grossly comparable to WT levels in Cre-negative mice.
These results indicate the utility of the Kcnma1 fl mouse line in further studies requiring tissue-specific deletion of the BK channel.