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J Physiol 589.6 (2011) pp 1489–1503
Functional and developmental expression of a zebrafish Kir1.1 (ROMK) potassium channel homologue Kcnj1 Leila Abbas1,2 , Saeed Hajihashemi1 , Lucy F. Stead3 , Gordon J. Cooper1 , Tracy L. Ware1 , Tim S. Munsey3 , Tanya T. Whitfield1,2 and Stanley J. White3,4 1
Department of Biomedical Science and 2 MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield S10 2TN, UK Institutes for Molecular and Cellular and Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK 4 Department of Physiology, Ross University School of Medicine, Commonwealth of Dominica, West Indies
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Non-technical summary Due to the conservation of developmental pathways and genetic material over the course of evolution, non-mammalian ‘model organisms’ such as the zebrafish embryo are emerging as valuable tools to explore causes and potential treatments for human diseases. Ion channels are proteins that form pores and help to establish and control electrical gradients by allowing the flow of ions across biological membranes. A diverse range of key physiological mechanisms in every organ in the body depends on the activity of ion channels. In this paper, we show that a potassium-selective channel that underlies salt reabsorption and potassium excretion in the human kidney is also expressed in zebrafish in cells that are important regulators of salt balance. Disruption of the channel’s expression in zebrafish leads to effects on the activity of the heart, consistent with a role for this channel in the control of potassium balance in the embryo. Abstract The zebrafish, Danio rerio, is emerging as an important model organism for the pathophysiological study of some human kidney diseases, but the sites of expression and physiological roles of a number of protein orthologues in the zebrafish nephron remain mostly undefined. Here we show that a zebrafish potassium channel is orthologous to the mammalian kidney potassium channel, ROMK. The cDNA (kcnj1) encodes a protein (Kcnj1) that when expressed in Xenopus laevis oocytes displayed pH- and Ba2+ -sensitive K+ -selective currents, but unlike the mammalian channel, was completely insensitive to the peptide inhibitor tertiapin-Q. In the pronephros, kcnj1 transcript expression was restricted to a distal region and overlapped with that of sodium–chloride cotransporter Nkcc, chloride channel ClC-Ka, and ClC-Ka/b accessory subunit Barttin, indicating the location of the diluting segment. In a subpopulation of surface cells, kcnj1 was coexpressed with the a1a.4 isoform of the Na+ /K+ -ATPase, identifying these cells as potential K+ secretory cells in this epithelium. At later stages of development, kcnj1 appeared in cells of the developing gill that also expressed the a1a.4 subunit. Morpholino antisense-mediated knockdown of kcnj1 was accompanied by transient tachycardia followed by bradycardia, effects consistent with alterations in extracellular K+ concentration in the embryo. Our findings indicate that Kcnj1 is expressed in cells associated with osmoregulation and acts as a K+ efflux pathway that is important in maintaining extracellular levels of K+ in the developing embryo. (Resubmitted 29 September 2010; accepted after revision 19 January 2011; first published online 24 January 2011) Corresponding author S. J. White: Department of Physiology, Ross University School of Medicine, Commonwealth of Dominica, West Indies. Email:
[email protected] Abbreviations ClC, chloride channel; hpf, hours post fertilization; Kir, inwardly rectifying potassium channel; MO, morpholino antisense oligonucleotide; NCCT, sodium–chloride cotransporter; Nkcc, sodium–potassium–chloride cotransporter; PDB, protein data bank; ROMK, renal outer medullary potassium channel; TAL, thick ascending limb of Henle’s loop; TEVC, two-electrode voltage clamp; TPNQ, tertiapin-Q.
L. Abbas and S. Hajihashemi contributed equally to this work. C 2011 The Authors. Journal compilation C 2011 The Physiological Society
DOI: 10.1113/jphysiol.2010.200295
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Introduction In mammals, the kidney is the major organ underlying K+ homeostasis, which is determined via regulated secretion of K+ by cells of the distal tubule and collecting duct. In addition, cells in the thick ascending limb of Henle’s loop (TAL) secrete potassium into the tubular lumen, a process that underpins both the primary dilution of tubular fluid and the concentration of the urine during water deprivation (Hebert et al. 2005). In the collecting duct, secretion of K+ determines urinary potassium excretion, but in the TAL, K+ secretion enables recycling of K+ between the cell and lumen, coupling the action of transporters and channels that underlie reabsorption of NaCl by this nephron segment (Wang et al. 1990). Potassium secretion in the distal nephron occurs predominantly via a class of ion channels known as inward rectifiers (Kir channels). Isoforms of the Kir1.1 (ROMK) family are expressed in the apical membranes of cells of the TAL, the distal and connecting tubule, as well as the cortical and outer medullary collecting duct (Xu et al. 1997). The zebrafish, Danio rerio, is a small freshwater teleost that has emerged as a powerful model of a number of human diseases and has great potential for studies of vertebrate integrative physiology (Briggs, 2002; Shin & Fishman, 2002). However, details of the functions of identified channels and transporters in ion transport by zebrafish embryonic renal tubules are largely unknown. The experiments described in this study were designed to determine the basic properties and sites of expression of a ROMK-like channel of the zebrafish as a first step in assessing the suitability of Danio as a model with which to investigate the pathogenesis of renal ion transport-related diseases of humans. Methods
J Physiol 589.6
(IMAGE) Consortium (No. 3815817) and was provided in the plasmid pME18S-FL3. The full length clone was sequenced and the open reading frame was subcloned into the vector pTLN (Lorenz et al. 1996). Sequence analysis
The genome sequence database of zebrafish (Ensembl genome, http://www.ensembl.org) was used for BLAST (basic local alignment search tool) searching. The deduced protein amino acid sequence and splicing information of kcnj1 was obtained from VEGA (http://vega.sanger.ac.uk/). Structural modelling
The query-template alignments were produced with Praline-TM alignment software built especially for transmembrane proteins (Pirovano et al. 2008). These alignments were then input to Modeller in PIR format. Structural models were created using Modeller 9v7 (Sali & Blundell, 1993) employing the ‘MyModel’ method to allow tetrameric proteins of the correct geometry (as indicated by the symmetry transformations within the template protein data bank (PDB) files) to be created. Both Kcnj1 and rat ROMK2 (rROMK2) models were based upon a Kir3.1-KirBAC1.3 chimera protein (Nishida et al. 2007), the structure of which has been solved by X-ray crystallography (PDB code 2qks) and has the highest sequence identity (35%) with both Kcnj1 and rROMK2. Three models were created of each channel. The lowest energy conformation of each channel was then selected to undergo further rounds of energy minimization via 200 cycles of steepest descent, performed with Swiss-pdbviewer, implementing the GROMOS 43B1 force field.
Work with animals
All experiments with Xenopus laevis and Danio rerio were carried out under the guidelines described by Drummond (2009) and approved by legislation and local institutional animal welfare committees as indicated. Cloning of kcnj1
A database search identified a partial clone in a zebrafish EST library with similarity to human Kir1.1 (kcnj1: Genbank Accession No. BF157829). In this study we follow the nomenclature guidelines for zebrafish genes (http://zfin.org/zf info/nomen.html), so that gene and transcript are all lowercase and italicized (i.e. kcnj1), and the protein not italicized, and with the first letter uppercase (i.e. Kcnj1). The clone was sourced from the Integrated Molecular Analysis of Genomes and their Expression
Electrophysiology
We determined the properties of macroscopic currents by conventional two-electrode voltage clamp (TEVC: Leipziger et al. 2000). Oocytes were obtained from mature female Xenopus laevis killed humanely using a procedure in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act of 1986. Briefly, frogs were immersed in anaesthetic 0.2% (w/v) tricaine methanesulfonate (supplemented with 5 mM Hepes, pH 7.80) until unconscious. The animals were then killed by decapitation and destruction of the spinal cord. Standard protocols were followed for the isolation and care of oocytes (Leipziger et al. 2000). For expression studies, defolliculated stage V/VI oocytes were injected (50 nl) with 1.6 ng of capped (mMESSAGE mMACHINE SP6 kit, Ambion) kcnj1 mRNA, 1 ng of rat C 2011 The Authors. Journal compilation C 2011 The Physiological Society
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Zebrafish K+ channel
ROMK2 mRNA (positive control), or an equal volume of water. Injected oocytes were incubated at 18◦ C in OR3 solution containing 6.85 g l−1 of Leibovitz L-15 cell culture medium plus 10,000 units ml−1 penicillin G sodium and 10,000 μg ml−1 streptomycin sulphate (Invitrogen), and 5 mM Hepes at pH 7.5. After 48–72 h, TEVC was performed using a Gene-Clamp 500B amplifier (Axon Instruments, Union City, CA, USA). Data were acquired using Clampex (pCLAMP, Axon Instruments, version 6) on an IBM compatible PC (Gateway) equipped with an analog-to-digital interface (Digidata 1200, Axon Instruments). The initial holding potential was maintained at −50 mV, and this was ramped from −120 to +100 mV in steps of 20 mV, and held for 50 ms at each test voltage and between each step voltage returning to –50 mV. For each recording this protocol was repeated 5 times and the signals averaged. Currents were determined mid-way through the time-independent phase of the current trace. Unless stated otherwise, experiments were performed at room temperature in amphibian Ringer solution (ND96) containing (in mM): NaCl (96), KCl (2), CaCl2 (1.8), MgCl2 (1), Hepes (5) at pH 7.5. Zebrafish embryos
The work with zebrafish described in this paper was carried out under UK Home Office Licence regulations and approved by the University of Sheffield Project Applications and Amendments Committee. Wild-type adult zebrafish (AB strain) were maintained on a 14 h light–10 h dark cycle and embryos obtained from mass matings. Developing embryos were maintained at 28.5◦ C in E3 medium, containing (in mM): NaCl (5), KCl (0.2), CaCl2 (0.3), MgCl2 (0.3), KH2 PO4 (0.05) and Na2 HPO4 (0.29), pH 7.0–7.2. Embryos were staged by standard criteria (Kimmel et al. 1995) in hours post fertilization (hpf). In situ hybridization of kcnj1 and other transcripts
Embryos were fixed in 4% paraformaldehyde/PBS for 3 h at room temperature and stored in methanol at −20◦ C. In situ hybridisation for kcnj1, the zebrafish orthologue of the renal chloride channel ClC-Ka (NM 200382), Na+ /K+ -ATPase a1a.4 (AY008376), Barttin (XM 001332980) and the thiazide-sensitive sodium chloride cotransporter NCC (NCCT) (NM 001045080) was carried out according to established protocols (Oxtoby & Jowett, 1993) using digoxigenin- or fluorescein-labelled RNA probes (all reagents from Roche). Double-labelled embryos were incubated overnight with anti-fluorescein-AP antibody 1:3000 in blocking solution at 4◦ C before washing and staining with Fast Red. The first antibody was C 2011 The Authors. Journal compilation C 2011 The Physiological Society
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inactivated with 0.1 M glycine-HCl, pH 2.2 in 0.1% Tween-20 at room temperature for 10 min. After washing, anti-digoxygenin-AP antibody 1:3000 was applied overnight and the embryos washed. The colour reaction was carried out in the dark in 4.5 μl ml−1 4-nitro blue tetrazolium chloride and 3.5 μl ml−1 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) in 0.1 M Tris pH 9.5, 50 mM MgCl2 , 0.1 M NaCl, and 0.1% Tween (PBTw). Single staining was performed essentially as above, omitting the fluorescein-specific steps. Staining reactions were quenched with PBTw washes followed by 2 h of 4% paraformaldehyde fixation and the embryos dehydrated through a glycerol series to 90% for mounting and photography.
Immunohistochemistry
Embryos were fixed in Dent’s solution (80% methanol, 20% DMSO) for 2 h at room temperature, followed by washing in PBTw and dehydration through a progressive (25% to 100%) methanol series. Following rehydration, embryos were blocked in 10% bovine serum albumin and incubated with a 1:1000 dilution of an anti-Nkcc monoclonal antibody (T4: Developmental Studies Hybridoma Bank, Iowa) overnight at 4◦ C. Samples were washed and incubated in a 1:200 dilution of anti-mouse peroxidase-conjugated secondary antibody (Sigma) overnight. Embryos were washed and the colour reaction developed using a diaminobenzidine (DAB) kit (Vector Labs) followed by a glycerol series for mounting and photography.
Morpholino antisense injections
Morpholino oligonucleotides (Gene Tools, LLC) were dissolved in water to a stock concentration of 10 mg ml−1 . Sequences were: kcnj1 morpholino: 5 -CTCTCTCAAGGAGCGAGTCATCTTA-3 ; mismatch control morpholino: 5 -CTgTCTgAAGGAcCGAcTCATgT TA-3 . Injections were carried out using a microinjection rig (Narishige) into the yolk of 1–8 cell stage wild-type embryos (0.5–2 nl per injection).
Heart rate measurements
Heart rates in beats per minute were determined in unanaesthetized 24 hpf and 48 hpf embryos by measuring the time (by stopwatch) taken for 15 ventricular contractions observed under a stereomicroscope. Determinations were performed in triplicate for each embryo.
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Data presentation and analysis
Unless otherwise indicated, data are presented as means ± S.E.M. Comparisons were made between means by ANOVA or Student’s unpaired t test as appropriate and values are presented as significant when P was
