Kcnq 1-5 (Kv7.1-5) potassium channel expression in the adult zebrafish
© Wu et al.; licensee BioMed Central Ltd. 2014
Received: 25 June 2013
Accepted: 11 February 2014
Published: 20 February 2014
KCNQx genes encode slowly activating-inactivating K+ channels, are linked to physiological signal transduction pathways, and mutations in them underlie diseases such as long QT syndrome (KCNQ 1), epilepsy in adults (KCNQ 2/3), benign familial neonatal convulsions in children (KCNQ 3), and hearing loss or tinnitus in humans (KCNQ 4, but not KCNQ 5). Identification of kcnqx potassium channel transcripts in zebrafish (Danio rerio) remains to be fully characterized although some genes have been mapped to the genome. Using zebrafish genome resources as the source of putative kcnq sequences, we investigated the expression of kcnq1-5 in heart, brain and ear tissues.
Overall expression of the kcnq x channel transcripts is similar to that found in mammals. We found that kcnq1 expression was highest in the heart, and also present in the ear and brain. kcnq2 was lowest in the heart, while kcnq3 was highly expressed in the brain, heart and ear. kcnq5 expression was highest in the ear. We analyzed zebrafish genomic clones containing putative kcnq4 sequences to identify transcripts and protein for this highly conserved member of the Kcnq channel family. The zebrafish appears to have two kcnq4 genes that produce distinct mRNA species in brain, ear, and heart tissues.
We conclude that the zebrafish is an attractive model for the study of the KCNQ (Kv7) superfamily of genes, and are important to processes involved in neuronal excitability, cardiac anomalies, epileptic seizures, and hearing loss or tinnitus.
Potassium channels are well-established biological targets for diseases including neuropathic pain, epilepsy, cardiac arrhythmia, hearing loss, deafness, or tinnitus. In particular, mutations in the KCNQ4 potassium gene and perhaps KCNQ3 are associated with progressive high frequency hearing loss[2, 3]. Of the several ion channels used by the sensory hair cell, the K+ channel KCNQ4 is thought to modulate the membrane potential of hair cells to adjust the sensitivity of hearing in a variety of mammals[1, 4, 5]. Similarly, KCNQ4 and KCNQ5 are key modulators of L-type Ca2+ channel activity in cardiovascular cells. Variants of KCNQ5 are not associated with sensory hearing loss in humans, but there is high abundance in the larval zebrafish ear[7, 8], and thus, may be related to yet to be defined developmental factors related to hearing.
A recent study characterized the expression of kcnq2, kcnq3, and kcnq5 in whole larval zebrafish (Danio rerio), but we know little about the expression of the complement of kcnq genes and the K+ ion channels that they encode in various organs of the adult zebrafish. Since certain drugs and metal ions affect the function of Kcnq channels[10–12] in a dose-dependent manner, these agents can be used to alter ion permeability across the membrane of zebrafish hair cells and thus create a fish model of sensory cell dysfunction. KCNQ2-5 channels are also regulated by intracellular signal transduction effectors such as phospholipids, phosphorylation, and calmodulin. However, little is known about how these signaling systems impact the kcnq channels in zebrafish sensory pathways. Thus, the zebrafish offers a unique opportunity to study Kcnq channel modulation, function and dysfunction.
The zebrafish has served as an especially attractive model for the study of the development and function of the vertebrate inner ear[8, 16]. It has three methods of sensing sound within its environment. The first involves the lateral line system, which is comprised of a set of neuromasts containing hair cells arrayed along each side of the body. Neuromasts contain bundles of sensory hair cells beneath a cupula, which are responsible for sensing the displacement of water molecules. The second means of sensing sound are structures of the inner ear composed of the utricle, saccule, lagena and pars neglecta. Each of these anatomical structures house patches of sensory hair cells and supporting cells that are embedded in the epithelial lining of the macula. The hair cells found in these structures are similar to those found in mammals, and contain voltage gated and ligand gated ion channels presumably linked to several signal transduction pathways. Third, there are sets of motion detectors or neuromasts arrayed around the head, particularly the orbital regions. In this report, we have studied Kcnq channel expression and localization in several tissues of the zebrafish. Using the deduced mRNA sequences in the available databases, we probed for the presence of Kcnq channel mRNA transcripts in the ear, brain and heart, and partially characterized the amino acid sequence of one channel protein. The zebrafish genome has two different kcnq4 genes, one of which has been localized to chromosome 19. The mRNA from this gene is also expressed in zebrafish brain and ear. We prepared a specific antibody to zebrafish Kcnq4, quantified its levels using qRT-PCR, and further verified its expression using Western blots of brain and ear tissues.
Detection of Kcnq Expression in Zebrafish
Summary of primers designed to amplify kcnq 1-5 and β-actin mRNAs
Expected size (bp)
5′- TCC AGT CGC TCA TGT GTC TC -3′
5′- TTT CAT CCC ACC TTC TTT GC -3′
5′- GAG CCA GTG CAG GAG AAA AG -3′
5′- TGA GGT AGA AGG CCG ACA CT -3′
5′- GAG AAG GAT TCG GCT CAC TG -3′
5′- GCG TCT GCA TAG GTG TCA AA -3′
KCNQ4 (a) fwd
5′-TAT GCA GAC TCC CTC TGG TG-3′
KCNQ4 (a) rev
5′-CCT GCA CTT TCA GAG CAA AG-3′
KCNQ4 (b) fwd
5′-GGG CCG CAG GGT TTC TTT AAA CTT-3′
KCNQ4 (b) rev
5′-ATG ACA GTA TGC TGC CGT CCT TCA-3′
KCNQ4 (c) fwd
5′-CGG CCG CAG GGT TTC TTT AAA CTT-3′
KCNQ4 (c) rev
5′-TCC TTC AGT GGG AAG ATG GGC TTT-3′
KCNQ4 ch19(a) fwd
5′-TGC CTG TAC AAT GTG CTG GAG AGA-3′
KCNQ4 ch19(a) rev
5′-AAG GCT TTC TGG CAA AGC GTA GTC-3′
KCNQ4 ch19(b) fwd
5′-ATC AGC CAA TGA TGA CAG ACG GGT-3′
KCNQ4 ch19(b) rev
5′-AAG GCT TTC TGG CAA AGC GTA GTC-3′
5′- TGC CTG GTA TAT TGG GTT CC -3′
5′- TGA ACC TTC AAG GCA AAA CC -3′
5′- TCC CCT TGT TCA CAA TAA CC- 3′
5′- TCT GTG GCT TTG GGA TTC A-3′
The inner ear tissue of zebrafish included the sensory epithelium (culled from 6 fish, both ears), consisting also portions of the utricle, saccule and lagena tissues, but not semicircular canals. Figure 1B shows mRNA expression of kcnq1-5 in the zebrafish ear - all kcnq transcripts were detected. However, kcnq1 was somewhat weak, while kcnq5b provided a much stronger signal. Figure 1C shows the expression pattern in zebrafish heart. Except for kcnq5b, transcripts for kcnq1-4 were detected.
qRTPCR of kcnq1-5 expression in brain, ear, or heart
Partial sequences of KCNQ4 obtained from PCR products
KCNQ4 (a) primers
-3′ (144 bp)
KCNQ4 (b) primers
KCNQ4 (c) primers
-3′ (358 bp)
Kcnq4 protein expression using Western Blots
Genetic and comparative analysis of Kcnq4 Proteins (channels)
Summary of kcnq genes in zebrafish (2013)
In this study, we characterized Kcnq-type proteins/channel expression in brain, heart, and ear tissues of the zebrafish. We show that members of the Kcnq (Kv7.x) family of mRNAs are present in these tissues. Further, we demonstrated mRNA as well as the protein for Kcnq4 in ear and brain extracts from adult zebrafish. Although signals for kcnq1 and kcnq5 were weak using end-point PCR, the transcripts were readily detected in all tissues using qRTPCR. These data are consistent with previous reports of the Kcnq1 channel expressed during development.
As previously found in mammals[1, 3–5, 23–25], kcnq2 was expressed in zebrafish brain, heart and ear. Similarly, in mammals, kcnq3 is usually found co-expressed in the same tissues. Kcnq4 was detected in ear and brain tissue using a Kcnq4 selective antibody. KCNQ4 is found in auditory hair cells in mammals and we suggest that it may be present in homologous cells in the zebrafish.
The amino acid sequences of zebrafish Kcnq4, as well as other members of the KCNQ channel family, are conserved across phylogeny. One distinguishing characteristic of KCNQ2 and KCNQ3 is the presence of a clustering domain that allows interaction of KCNQ channels with Na+ channels in the nodes of Ranvier. Another characteristic of KCNQ channels is that the structural assembly (homotetramer vs. heterotetramer) is dependent upon amino acid sequences in the carboxyl-terminal region. In the case of zebrafish Kcnq4, the translated amino acid sequence that we derived (Figure 4A) is consistent with the head-linker-tail structure of KCNQ4 that supports a homotetrameric structure.
The more highly abundant KCNQ transcripts expressed in the brain (KCNQ2, KCNQ3, and KCNQ5) are possible contributors to a number of important electrophysiological functions that are necessary for normal cognitive function. That is, dysfunction of these channels has been associated with dementia, stroke, and epilepsy. Very similar to the mammalian cochlea[4, 25], but perhaps more similar to the vestibular system, our results show that the zebrafish inner ear sensory tissues do express the kcnq 2-5 genes. The inhibition of KCNQ4 activity in the mammalian cochlea or knockout mouse causes sensory cell degeneration followed by deafness. However, unlike mammals, the zebrafish hair cells are capable of regeneration after acoustic or chemical insult[30, 31], and selected transcription factors among other putative molecules are key mediators of the regeneration[32, 33]. No variants of KCNQ5 are associated with sensory hearing loss in humans so perhaps its high abundance in the zebrafish ear is associated with regenerative capabilities.
Studies of the effects of exogenous regulators of zebrafish hair cell regeneration are at various stages of investigation. Our identification of Kcnq channels in zebrafish may offer a new in vivo model system for screening KCNQ channel modulators/drugs and their effects on regeneration. Certain classes of drugs are being designed to modulate the activity of specific KCNQ-type channels[35–37], and our work suggests that screening this class of chemotherapeutic agents for functional[38–40], as well as for adverse effects (such as behavioral abnormalities) in the zebrafish is promising. Further, expression of the channels cloned from the zebrafish in heterologous systems[15, 41, 42] provides an attractive platform for electrophysiological studies since dissociated hair cells from the inner ear of the zebrafish are extremely difficult to patch (Moore, unpublished observations, 2010; however, see).
Recent advances in sequencing the zebrafish genome have provided further insight into modeling human diseases[44, 45]. Nevertheless, the chromosomal localizations and/or complete sequencing of the kcnq4 gene remain to be completed. Western blots demonstrated that the Kcnq4 protein is expressed in the brain as well as the ear. Thus, using the zebrafish with its rapid developmental period as a laboratory specimen may accelerate genetic screening for more specific KCNQ channel mutants, and perhaps foster drug discovery strategies for chemotherapeutic intervention in diseases associated with mutations in the Kv (x) family of genes, e.g., conditions manifested in humans such as hearing loss, and tinnitus.
Animal procedures were approved by the NU-ACUC (Approval number 2006 - 1034) and were performed in accordance with regulations for the care and use of laboratory animals. Adult zebrafish (initial stock was a kind gift from Dr. Jacek Topczewski, Ann Lurie Children’s Research Medical Center, Northwestern University, Chicago, IL) were kept in an aquarium that was maintained at 25°C, filtrated, pH balanced, with frequent removal of excess nitrate, nitrite, ammonia, chloramines, and chloride. Exchange of conditioned tap water occurred at regular intervals. Two bottom feeder fish (Bristle nose catfish, Ancistrus temmincki) were kept in the aquarium to reduce the accumulation of waste. Animals were fed twice daily using a combination of flake or morsels that had been sterilized (UV illumination overnight) before usage. Wild type embryos were collected from natural matings in our lab, or ordered from ZIRC (University of Oregon, Eugene, OR), and were kept in 12-well clusters (~n = 6 each well) at 28.5°C in an air-only incubator. Stages were referred to in hours post-fertilization (hpf) or days post-fertilization (dpf). After 24-hpf, some larvae were maintained in 0.03% phenylthiourea to prevent melanin pigment formation to ease the identification of a normally developed lateral line.
Zebrafish were sacrificed using a combination of Tricaine Methylsulphonate (MS-222) and ice, and the various tissues were rapidly removed. Zebrafish brain, heart, and ear were dissected, used immediately for experimentation, or pooled separately in 1.5 ml Cryovials and placed in liquid nitrogen until use. Total RNA was extracted from the tissues using TRIzol reagent (Invitrogen, Carlsbad, CA). Ear tissues were placed in 6.0 ml of low calcium saline (LCS, 10 mM HEPES, 100 μM CaCl2, 110 mM NaCl, 2.0 mM KCl, 2.0 mM MgCl2, 3.0 mM D-glucose, pH 7.3). EDTA and MgCl2 (12 μl) were added to the ear tissues and incubated for 15 min to prevent calcium carbonate leakage from the inner ear otolithic structures.
RT-PCR and PCR
Primers for kcnq1-5 and controls were designed based on zebrafish DNA sequences found in publically available databases such as the NCBI (GenBank), and Ensembl. The nucleotide sequence was searched using BLASTN to determine the number and location of different exons. Primers were designed using PrimerQuest (IDT, Coralville, IA). Nested primers were designed to cross exon boundaries and selected to amplify a 200 – 600 bp fragment of the desired kcnq mRNA. Primers were selected for optimum base content and annealing properties to the desired mRNA. Searching the zebrafish genome and expressed mRNAs was conducted using the BLAST suite of programs.
Total RNA pellets were washed with 75% ethanol, dried, suspended in RNAse free water and stored at a temperature of 4°C. The RNA concentration and purity was quantified by spectrophotometry (Beckman DU-7500, Fullerton, CA) using the absorbance ratio of A260/A280. Individual PCR reactions contained forward and reverse primers for the desired target, and control reactions contained primers for β-actin. The one-step RT-PCR system (Invitrogen, Carlsbad, CA) was used for amplification of target mRNAs. For each reaction, 200 - 400 ng of total RNA was used for the RT-PCR.
Reactions were performed in a thermocycler for 30 cycles (Techne, TC-312, Minneapolis, MN) with recommended denaturation (94°C, 2 min), annealing (55°C, 30 s), extension (72°C, 2 min), and hold (72°C, forever). The PCR products were separated using 1.0 – 2.0% agarose gel electrophoresis with gels containing ethidium bromide. Gel photographs were taken (Kodak, DC 290, Rochester, NY), transferred and stored to a microcomputer (Dell Dimension 8200). The molecular ladder (M) of the gels was separated by 100 bp bands with the first band at the bottom being 100 bp; the brightest band near the top of the ladder is at 600 bp.
Sequencing of kcnq4 PCR products
Gel bands were excised and purified using purification columns (Catalog #K2100, Invitrogen, Carlsbad, CA) designed for agarose gel extracts. The purified DNA products were sequenced at Sequetech (Mountain View, CA). The 5′ PCR primers for each product were used as sequencing primers (see Table 1).
Quantitative RTPCR (qRTPCR)
Sequences of primers used for qRTPCR
Sequence 5′ – 3′
TTC ACA GGG CCA TCT CAA CCT CAT
TCA AGC GCT CTG AAC TTG TCT GGA
TCA GCG GAT TCA GCA TCT CAC AGT
TGT CCG ATT CAC CCT CTG CAA TGT
AAC TCC ATT TCC GTA CCC ATC CCA
TGT CTC TCC ACC CGC ACA AAT CTA
TTT CGC ACA TCT CTG CGC CTC AAA
TCC ATG GCC ACG TCA CAG TAA GAT
ACA ACC AAC CTT CCA GTC CAG ACA
TAA CTC ACT AAA CCG CTG GTG GCT
TTG CCG TTC ATC CAT CTT TGA CGC
TCA GGT CAC ATA CAC GGT TGC TGT
A synthetic peptide CSGKMGFRDRIRMNNSRSS based upon the reported partial cDNA, and putative amino acid sequences for zebrafish Kcnq4 was prepared and conjugated to Kehoe Limpet hemocyanin (KLH) for immunization. Two rabbits were used for antibody production by Bio-Synthesis (Lewisberg, TX). Antisera were obtained 6 -10 weeks after immunization and characterized for immunoreactivity against zebrafish tissue extracts.
Zebrafish brain and ear were homogenized in a lysis buffer (20 mM Tris, 150 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 2.5 mM NaPyrophosphate, 1.0 mM Na Vanadate, 1.0 mM betaglycerol phosphate, 1.0 mg/mL leupeptin, and 1.0% Triton X-100, pH 7.5) using a motor driven pestle (5 bursts of 10 s) on ice. The crude homogenate was centrifuged at 1.4 × 103 rpm (Eppendorf microfuge, Hauppauge, NY). The supernatant fraction was removed, saved and the pellet extracted again using lysis buffer plus 1.0% SDS. After centrifugation to remove insoluble material, the soluble pellet fraction was saved. Protein concentrations in each fraction were determined using a reagent (Pierce BCA, Pittsburgh, PA) as suggested by the manufacturer. Electrophoresis was conducted on 4 - 12% SDS Page minigels (Invitrogen, Grand Island, NY). A total of 25 - 30 μg of brain fractions, ear and heart fractions (10 - 12 μg) were loaded into separate lanes on the gel. After electrophoresis, the gel was blotted to PVDF membranes (Millipore, Billerica, MA) using a Biorad transfer cell and Tris-Glycine-20% methanol transfer buffer. Transfer was accomplished at a constant voltage (40 V) for 1.5 hrs at room temperature. Membranes were blocked in 5.0% nonfat dry milk in Tris-buffered saline (20 mM Tris -150 mM NaCl, pH 7.5) containing 0.1% Tween 20 (TBS-T). Blots were then treated with anti-Kcnq4 peptide antiserum (1:2000) in TBS-T with 5.0% nonfat dry milk at room temperature for 2.0 hrs. After washing 5x with TBS-T, the blots were then incubated with HRP-conjugated Goat anti-rabbit antibody (Bio-Rad, 1:5000) for 1.0 hr at room temperature. After washing 5x with TBS-T, the blot was developed with electrochemiluminescent substrate (Pierce West Pico, Pittsburgh, PA) and bands were detected on film (Kodak Biomax, Rochester, NY).
Expression intensity of gene transcripts was analyzed using Image J (NIH Image, Bethesda, MD). Data display was accomplished using Origin (v8.0 Origin Software, Northampton, MA).
We are indebted to Allie Coffin for teaching us how to dissect the zebrafish inner ear. We thank Arthur Popper for introducing us to Allie. We received financial support from the Alliances for Graduate Education in the Professoriate (AGEP) (KS - EJM), American Society of Pharmacology & Experimental Therapeutics (ASPET) (KL - EJM), the Summer Research Opportunities Program (SROP) (MH, CT - EJM) of the Graduate School at Northwestern University, the Montel Williams MS Foundation (EJM), and a UNT Research Opportunities Program grant (CW - EJM). We thank Nicole Calderon and Daniel Ledee for assistance with certain of the RT-PCR protocols.
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