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Translational Physiology
1 Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan
2 Laboratory of Veterinary Pathology, Osaka Prefecture University, Sakai, Japan
3 Safety Research Laboratory, Tanabe Seiyaku Company, Limited, Osaka, Japan
4 Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan
5 Nagisa Hospital, Hirakata, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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voltage-dependent potassium channel; deafness; long-QT syndrome; achlorhydria; hypertension
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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) subunit of the voltage-gated K+ channel. It encodes six membrane-spanning domains (S1–S6), including the voltage sensor (S4) domain, and a K+-selective pore between S5 and S6 (2, 24). To form native channels, KCNQ1 coassembles with small ß-subunits, so-called KCNE proteins. Although the stoichiometry of coassembly is not yet known, it is likely that four -subunits assemble with four ß-subunits to form the channels. In the heart, KCNQ1 is coassembled with KCNE1. The KCNQ1/KCNE1 complex produces a slowly activating delayed rectifier K+ current (IKs) that contributes to the later phase of action potential repolarization, returning to the resting potential (2, 24). Mutations of the human KCNQ1 gene are associated with the congenital long-QT syndrome, an inherited disorder that is characterized by abnormal ventricular repolarization and increases the risk of sudden death from cardiac arrhythmias. There are at least two familial forms of long-QT syndrome. One is the Jervell and Lange-Nielsen syndrome, which is believed to be inherited as an autosomal recessive trait and associated with congenital deafness (12). A second, more common familial form is inherited as an autosomal dominant trait with no other phenotypic abnormalities. This form, which is sometimes referred to as the Romano-Ward syndrome (34), is usually associated with a lower arrhythmia risk than the autosomal recessive form.
In the inner ear, both KCNQ1 and KCNE1 are expressed. In this tissue, the KCNQ1/KCNE1 complex produces a K+-rich fluid known as endolymph that bathes the organ of Corti, the cochlear organ responsible for hearing, and the utricle, saccule, and semicircular canal, which are responsible for balance and equilibrium. Functional loss of KCNQ1 provokes congenital deafness in the individuals with Jervell and Lange-Nielsen syndrome. The mechanism of deafness is that the lack of IKs leads to inadequate endolymph production and deterioration of the organ of Corti (31).
In the stomach, KCNQ1 and KCNE2 are both expressed in the luminal membrane of the acid-secreting parietal cells (6, 11), where H+/K+-ATPase, a pump responsible for the transfer of H+ ions into the stomach, is coexpressed (9). The KCNQ1/KCNE2 complex yields K+ currents that are activated by acidic pH stimulation at resting membrane potential. Thus KCNQ1/KCNE2 forms acid-activated luminal K+ channels whose function is to supply K+ to the luminal surface to allow H+-for-K+ exchange by the pump (11). Disruption of Kcnq1 in mice causes a large increase in stomach pH that is accompanied by gastric hyperplasia (16).
In the small intestine and colon, KCNQ1 colocalizes with KCNE3 in the basolateral membranes of crypt cells (6). The KCNQ1/KCNE3 complex is constitutively opened at the more negative membrane potential of intestinal epithelial cells and can be further activated by the action of cAMP (25). This complex is thought to be important for maintenance of transepithelial transport in the colon and the small intestine, by recycling K+ that is transported into the cell by basolateral Na+-K+-2Cl– (NaK2Cl) cotransporters and Na+/K+-ATPases. Thus the KCNQ1/KCNE3 channel is required for transepithelial cAMP-stimulated Cl– secretion.
In the airway epithelia, KCNQ1 mediates a basolateral K+ conductance that plays an important role in maintaining cAMP-dependent Cl– secretion (10, 17). The ß-subunit coassembled with KCNQ1 in the airway epithelia has been a matter of debate. In the mouse, KCNE3 is thought to be coupled with KCNQ1 (10). In the pancreas, KCNQ1 is expressed in the insulin-secreting cells. Inhibitors for the KCNQ1 channels increase the insulin secretion, which suggested that KCNQ1 would be involved in insulin secretion by the regulation of membrane potentials in the insulin-secreting cells (29).
Kcnq1 expression is not only confined to the tissues described above but also occurs in many epithelial tissues, such as placenta, kidney, liver, and thymus (7, 33, 38). However, no clear functional role of Kcnq1 in these tissues has been found as yet. For a further understanding of Kcnq1 functions in these tissues as well as the establishment of novel therapeutic procedures for diseases associated with Kcnq1 dysfunction, such as the long-QT syndrome, deafness, and achlorhydria, an animal model that is easy to investigate and manipulate is required. To address this issue, a rat model would be suitable, since the rat has an 
10 times larger size than the mouse and offers several unique advantages in various fields of research. In particular, the rat has been used as a variety of disease models because of its easier clinical examination and sampling as well as therapeutic administration and manipulation.
In this report, we describe the characteristics of a novel Kcnq1 mutation, deafness Kyoto (dfk), in the rat. Positional candidate cloning revealed that the dfk allele is an intragenic deletion including exon 7 of the Kcnq1 gene. WTC-dfk rats suffer from deafness and imbalance resulting from profound morphological abnormalities of the inner ear. These rats exhibit prolonged QT intervals and T-wave abnormalities on electrocardiogram (ECG) measurements and elevation of pH to almost neutral in the gastric secretion. Additionally, WTC-dfk rats display hypertension.
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MATERIALS AND METHODS |
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The ACI/NKyo (NBRP no. 0001), WTC (NBRP no. 0020), and WTC-dfk (NBRP no. 0289) strains have been deposited in the National BioResource Project for the Rat in Japan and can be obtained from the Project (http://www.anim.med.kyoto-u.ac.jp/nbr/).
Auditory brainstem response measurement.
Auditory brainstem response (ABR) measurements were performed in three individuals each for WTC and WTC-dfk rats at 26 wk of age. The following experiments were done on animals that were anesthetized with ketamine hydrochloride (60 mg/kg, im) and pentobarbital sodium (21 mg/kg, ip) and kept at 38°C. Stainless steel needle electrodes were inserted subcutaneously into the vertex (indifferent) and one side (active) and the other side (ground) of the retroauricular region, respectively. The ABR was obtained by averaging 1,000 evoked responses to click stimuli at intensities of 60, 65, 70, 75, 80, 100, 120, and 135 dB peak equivalent sound pressure level (peSPL), with 50-ms intervals generated by an acoustic stimulator (MEB-5504; Nihon Koden, Tokyo, Japan). Clicks were delivered through an inner ear-type earphone facing the meatus acusticus externus.
Genetic mapping.
(WTC-dfk x ACI)F1 rats were intercrossed to obtain F2 rats. Homozygous WTC-dfk animals were identified on the basis of appearance of head-tossing and/or circling behaviors and inability to swim at 3–4 wk of age. Two hundred and forty WTC-dfk animals were obtained from 1,000 F2 progeny and used for genetic mapping. For the initial mapping of dfk, we employed pooled simple sequence-length polymorphism (SSLP) analysis (27). DNAs from 45 randomly selected rats were standardized to 20 ng/µl, and equal amounts of individual DNAs were pooled. The dfk DNA pool was genotyped for 75 microsatellite markers distributed among all autosomes. For the fine mapping of dfk, all dfk/dfk animals were genotyped.
Sequencing.
PCR products were treated with ExoSAP-IT (Amersham Biosciences, Piscataway, NJ) to digest single-strand DNAs and excess primers. Cycle sequencing was performed with the BigDye Terminator Ready Reaction Mix according to the manufacturer's instructions (Applied Biosystems, Fater City, CA). PCR samples were purified with CENTRI-SEP spin columns and were then loaded into an ABI PRISM 3100 genetic analyzer (Applied Biosystems).
Northern blotting.
Poly(A) RNA was purified from total RNA by using Oligotex-dT30 Super (Roche Diagnostics). Two micrograms of poly-A RNA were electrophoresed on formaldehyde-denaturing agarose gels and blotted onto Hybond N+ membrane (Amersham Biosciences). Bands on the autoradiograph were detected by using an imaging plate and a computerized image display system (BAS2000; Fuji Film, Tokyo, Japan). A DNA fragment containing exons 9–14 of rat Kcnq1 was used as a probe.
Electrocardiograms.
ECG recordings were obtained from adult (12–18 wk) wild-type (n = 6) and WTC-dfk (n = 6) rats using implantable Physiotel TA10EA-F20 radio frequency transmitters and receivers (Data Sciences International, Arden Hills, MN). After an animal was anesthetized with chloral hydrate (600 mg/kg, ip), the transmitter was placed within the peritoneal cavity. The electrodes were placed at the right axilla and at the left side of the xiphoid. Each electrode was sutured subcutaneously. After implantation of the transmitters, the animals were allowed to recover for at least 72 h. Twenty consecutive beats were recorded from individual animals under resting conditions. Unfiltered data were analyzed offline, and QRS, QT, PQ, and RR intervals were measured. Rate-corrected QT values (QTc) were derived using the formula QTc = QT/SQRT(RR/100) (19).
Histopathological analyses.
To evaluate the histological phenotype of dfk rats, WTC-dfk rats were killed under anesthesia at 34 wk of age (n = 3). Age-matched WTC rats (n = 3) were examined as controls. Perfusion fixation through the left ventricle was performed with 4% paraformaldehyde in 0.1 M phosphate buffer. Brains, spinal cords, inner ears, stomach, and representative visceral organs were removed and fixed with the same fixative solution. Before processing for paraffin embedding, the ear tissues were decalcified with 5% EDTA-dipotassium salt. The samples were embedded in paraffin, and sections (4 µm) were stained with hematoxylin and eosin stain (HE). The cochlea was also embedded in epoxy resin, and then semithin sections (1 µm) were stained with toluidine blue.
Stomach pH and acid output measurements.
The WTC-dfk rats (11 wk old, n = 5) and the age-matched, wild-type, control WTC rats (n = 5) were fasted overnight before the experiment with free access to water. Each rat was anesthetized, and the abdominal cavity was opened and the pylorus ligated. Four hours after treatment, the rat was killed and the stomach was removed. The intraluminal contents were drained into a centrifuge tube after a small nick was made in the stomach wall along the greater curvature adjacent to the pyloric ligature. The solutions with the stomach contents were centrifuged to pellet the insoluble material. The pH of the supernatants was measured, and then acid was titrated using an automatic titrator COMTITE-500 (Hiranuma Sangyo, Ibaraki, Japan). The results were expressed as microequivalents of acid per liter of gastric volume.
Blood pressure measurement.
Systolic blood pressure was measured by the tail-cuff method using a nonpreheated, noninvasive blood pressure monometer (MK-2000; Muromachi Kikai, Tokyo, Japan). The average values of three measurements were determined for individual animals. All measurements were performed on 11 male rats at 9 or 10 wk of age (18).
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RESULTS |
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The adult WTC-dfk rats seemed to be smaller than the normal littermates, so we measured the body weights of the WTC-dfk rats (male, n = 6) at 5, 6, and 10 wk of age. At 5 or 6 wk of age, body weights were not significantly different between WTC and WTC-dfk rats. At 10 wk of age, WTC-dfk rats showed significantly lower body weight than WTC rats (246.3 ± 3.2 vs. 190.6 ± 8.8 g, P < 0.01) (Fig. 1A) .
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Identification of the dfk mutation.
The pooled SSLP analysis showed a linkage relationship between D1Rat429 and the dfk locus. A genetic linkage study of 240 WTC-dfk rats using 19 additional markers on Chr 1 narrowed down dfk to a 0.8-cM interval between D1Kyo9 and D1Mgh10 (Fig. 2, A and B). The dfk gene showed no recombination with D1Wox9 in 480 informative meioses. Within the dfk locus, four genes, Cd81 (CD 81 antigen), Kcnq1 (potassium voltage-gated channel, subfamily Q, member 1), Mrgrg (Mas-related G-protein coupled receptor member G), and Dhcr7 (7-dehydrocholesterol reductase), have been mapped, and these genes were thought to be candidates for dfk.
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To verify and diagnose the deletion at the molecular level, we designed the PCR primers rKcnq1–51 (5'-ACCTGTCATGGCTCCCTAGA-3') and rKcnq1–42 (5'-AGGCTGTCCTCAGCAAGAAG-3'), which are located outside of the 5'- and 3'-breakpoints, respectively (Fig. 2B). These primers yielded a 695-bp PCR product from WTC-dfk and a 2,735-bp PCR product from the wild-type WTC (Fig. 2E).
Histopathology of the inner ear.
To identify the histopathological alterations responsible for the deafness and imbalance observed in WTC-dfk rats, we examined the inner ear structures. In the cochlea of the 34-wk-old WTC-dfk rats, the Reissner's membrane was collapsed and the volume of the cochlear duct was markedly reduced. The stria vascularis was severely atrophied. The inner and outer hair cells in the Corti were degenerated, and swelling of supporting cells was observed. The number of neurons in the spiral ganglion was markedly reduced (Fig. 3B). In the macula statica of the utricle, the membranous labyrinth was collapsed onto the macula. The statoconia, the statoconial membrane, and the hair cells were compressed by the collapsed membranous labyrinth, and the endolymphatic space was not observed. The hair cells and supporting cells were severely disarrayed (Fig. 3D). In the ampullary crest of the semicircular ducts, the membranous labyrinth was also collapsed and the endolymphatic space was dramatically reduced. The collapsed membranous labyrinth compressed the sensory hair cells and the KCNQ1-expressing dark cells. Vacuoles were observed in the hair cells located in the marginal region of the ampullary crest (Fig. 3F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We found several intriguing phenotypes of the WTC-dfk rat. First, these rats showed circular movements, imbalance, and complete deafness. Histological analyses demonstrated the marked reduction of the endolymph of the inner ear and the collapse of sensory hair cells. These findings implied that endolymph reduction followed by hair cell collapses resulted in inner ear defects in the WTC-dfk rats. Kcnq1-deficient mice also show circular movements, imbalance, deafness, and inner ear pathologies that are very similar to those observed in the WTC-dfk rats (5, 16). The phenotypic similarities and deduced functions of the mutated KCNQ1 of the WTC-dfk rat suggested that WTC-dfk rats would be deficient for KCNQ1 protein.
Second, the WTC-dfk rats manifested prolonged QT interval and abnormal T-wave form in the ECG, indicating that the ventricular repolarization was prolonged in the WTC-dfk rats. The ventricular repolarization derives from transient outward K+ currents (Ito) and delayed, outwardly rectifying K+ currents (IK) (21). In the rat myocytes, two transient outward K+ currents, Ito,f and Ito,s (1, 36), and five distinct delayed rectifier currents, IKr, IKur, IK, IK,slow, and Iss, have been identified (1, 3, 20). However, IKs current generated from KCNQ1/KCNE1 complex is not a prominent repolarizing current in adult rats or in mice. Therefore, the cardiac phenotype seen in the WTC-dfk rats would not be a direct effect of KCNQ1 deficiency but would be induced by extacardiac stimuli such as autonomic nerve and hormonal factors (32, 35). This idea is supported by the data on the cardiac phenotypes of the Kcnq1-deficient mice. They displayed abnormal T-wave form and prolongation of the QT interval when measured in vivo, but not in isolated hearts (5). In addition, nicotine challenge of the isolated heart and acute stress due to saline injection in vivo revealed that sympathetic stimulation induced a long-QT phenotype in Kcnq1-deficient mice (28).
Third, we found achlorhydria in the WTC-dfk rats. Because gastric H+ is secreted by the H+/K+-ATPase with coupling to the uptake of the luminal K+ (23, 37), it is possible that the mutated KCNQ1 polypeptide fails to transport K+ into the gastric lumen. Histological findings of the WTC-dfk stomach were hypertrophy of the gastric glands and fibrosis in the lamina propria. Because the administration of proton pump inhibitor sometimes induces hypertrophic gastric glands in the rat, these pathological changes are thought to have been produced as a direct effect of the elevation of pH in the stomach (4). Kcnq1-deficient mice also show achlorhydria and increased weight of the stomach resulting from mucous neck cell hyperplasia (8, 16).
It is likely that the lower body weight found in the WTC-dfk rats is related to their hyperactivity associated with the inner ear defects. Additionally, the loss of Kcnq1 functions in the stomach, the intestine, or the pancreas might give another possible explanation. Gastric H+ is required to converse pepsinogen to pepsin, the protease largely responsible for initiating the digestion of proteins in the stomach. KCNQ1 expressed in the epithelium of the small intestine and colon is believed to regulate K+ transport into the lumen (25). Luminal K+ is required for transepithelial Cl– secretion, which regulates the osmolality of the intestinal or colonic mucus. In the insulin-secreting cells, the KCNQ1 channels might play a role in regulation of the insulin secretion (29). Thus investigating the digestive capability and insulin secretion levels in the WTC-dfk rats would be helpful for determining the cause of the lower body weight.
Lastly, the WTC-dfk rats displayed hypertension. This is the first evidence that Kcnq1 might be involved in the regulation of blood pressure in the rat. Hypertension is provoked by a variety of etiologies. We have not yet determined which factor(s) induces hypertension in the WTC-dfk rats, but it seems likely that it is due to a defect in reabsorption in the proximal tubule of the kidney. In the mouse kidney, KCNQ1 colocalizes with KCNE1 in the brush border of the mid to late proximal convoluted tubule as well as in the proximal straight tubule (30). It is thought that the KCNQ1/KCNE1 complex would make K+ flux to the lumen, which is essential to counteract membrane depolarization due to electrogenic Na+-coupled transport (30). Considering the important role of reabsorption in the regulation of blood pressure, it is possible that there might be some defect(s) in the kidney proximal tubule of the WTC-dfk rats.
WTC-dfk rats offer a sophisticated genetic system for studies of the physiological functions of KCNQ1, because this strain has a strict control strain, WTC. The two strains are coisogenic and have an identical genetic background except for the dfk deletion. Thus phenotypic differences found between them would only result from the dfk mutation, which would imply the involvement of the Kcnq1 in such phenotypes. This genetic system was obtained simply by a spontaneous mutation arising in an inbred strain. In the gene knockout mouse, one cannot exclude effects of genes closely linked to the targeted gene on their phenotypes, even after producing congenic strains (26). Kcnq1 has been shown to be associated with several diseases such as long QT, deafness, achlorhydria, and hypertension. To develop more effective treatments for these disorders, WTC-dfk could offer a powerful new tool as a Kcnq1-related disease model. Furthermore, because KCNQ1 is expressed in various epithelial tissues, including lung, colon, small intestine, and thymus (7, 33), the functions of KCNQ1 in these tissues could be clarified using the coisogenic system of WTC-dfk and WTC.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of K. Kitada: Laboratory of Mammalian Genetics, Center for Advanced Science and Technology, Hokkaido University, Kita 10 Nishi 8, Kita-ku, 060-0810 Sapporo, Japan.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Serikawa, Institute of Laboratory Animals, Graduate School of Medicine, Kyoto Univ., Sakyo-ku, Kyoto 606-8501, Japan (e-mail: serikawa{at}anim.med.kyoto-u.ac.jp)
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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