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Molecular Identi¢Cation and Characterisation of the Human Eag2

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Molecular Identi¢Cation and Characterisation of the Human Eag2 FEBS Letters 524 (2002) 204^210 FEBS 26330 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Molecular identi¢cation and characterisation of the humanprovided eag2 by Elsevier - Publisher Connector potassium channel M. Ju, D. Wrayà School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK Received 27 May 2002; revised 25June 2002; accepted 28 June 2002 First published online 9 July 2002 Edited by Maurice Montal without inactivation; the (slow) activation kinetics depend on Abstract We report the molecular cloning from foetal brain of the human potassium channel heag2. The cDNA encodes a pro- holding potential (‘Cole^Moore shift’) and extracellular mag- tein of 988 amino acids, 73%identical to heag1. Heag2 is ex- nesium, while the channel can be blocked by intracellular pressed in the brain, but is also found in a range of tissues calcium. The rat eag channels are expressed predominantly including skeletal muscle. In oocytes, the channel is a non-in- in neural tissue; cell cycle-speci¢c expression of eag1 has activating outward recti¢er, with dependence of activation rate been demonstrated that may be of great importance in cellular on holding potential. Compared with heag1, the conductance^ development and tumour growth. Finally, members of the elk voltage curve for heag2 was shifted to the left, the voltage subfamily [3] show electrophysiological features like eag (e.g. sensitivity was less, activation kinetics were di¡erent, and the elk1), or features intermediate between eag and erg (e.g. elk2), sensitivity to terfenadine was lower. The heag2 channel may though they do not show the Cole^Moore shift [3]. have important physiological roles. ß 2002 Published by Else- Here, we report the molecular cloning and characterisation vier Science B.V. on behalf of the Federation of European Bio- chemical Societies. of the human eag2 (heag2) channel, and we have compared its functional properties with the previously cloned [6] heag1 Key words: Potassium channel; Human sequence; channel. Outward recti¢er Kþ channel 2. Materials and methods 2.1. Cloning of heag2 1. Introduction Predictions for the sequence of the human eag2 channel were obtained using the sequence for the rat eag2 channel [4,5] in BLAST Potassium channels have key roles in setting membrane searches against the human genome, together with subsequent analysis potentials, action potential repolarisation and neuronal ¢ring using Genscan. From this, primers were designed as follows: frequency. Consequently they are of vital importance in neu- 5P-atgccggggggcaagagagggct (sense), 5P-atcctgacatggtatctgggggggtac (antisense) or 5P-ggcaggccagaatccagctggacatg (antisense). These prim- rotransmitter and hormonal release, cardiac function, as well ers were used in PCR reactions using a human foetal brain cDNA as cell cycling and development. These channels are also of library (Clontech), and products were cloned into pGEM-TEasy. Sev- great importance as sites of drug action, and are the sites of eral clones were sequenced (four completely, two partially) to check numerous pathologies such as inherited disorders [1]. Potassi- for sequence consistency. Heag2 and heag1 clones (the latter in pcDNA3, accession number um channels comprise several families [2]: two-transmem- AJ001366 [6], gift of L. Bernheim) were subcloned into pGEMHE brane inward recti¢ers, four-transmembrane ‘twin-pore’ chan- (gift of O. Pongs) for expression in oocytes using EcoRI or BamHI nels, and six-transmembrane voltage-dependent channels respectively, and a Kozak sequence inserted into heag2. After linear- which include Shaker, KvLQT and ether-a-go-go channel isation with NotI, capped cRNA was transcribed in vitro using the T7 families. promoter (Ambion MEGASCRIPT). The ether-a-go-go family is characterised by long N- and C- 2.2. Tissue expression studies terminal intracellular tails, and is subdivided into eag, elk and A probe was constructed by PCR using bases 2215^2423 of the erg subfamilies. Within this family, there are now known to be heag2 sequence (probe A) in the variable domain, sequenced, and a total of eight genes in rat [2,3]: eag1, eag2, elk1, elk2, elk3, labelled with digoxigenin-11^dUTP using the random primer method (Roche). The probe was used to screen a multiple-tissue Northern blot erg1, erg2, erg3. The last member to be cloned, rat eag2, was (MTN, Clontech), and labelling detected by anti-digoxigenin antibody obtained only recently [4,5]. conjugated to alkaline phosphatase, with detection by luminescent The erg subfamily [3] is characterised by fast activation, light emission using CSPD (Roche). very fast inactivation and inward recti¢cation. Erg channels For RT-PCR expression studies, a multiple-tissue cDNA panel are widely expressed, including the brain and heart, where (MTC, Clontech) was used in PCR reactions, using primers designed to amplify bases 2513^3125 of the heag2 sequence using heag2-speci¢c cardiac herg1 underlies the IKr current in the heart and its primers. The heag2 clone was used as positive control, and primers dysfunction can lead to dangerous arrhythmias. In contrast, against G3PDH were used to check standardisation of cDNA quanti- the eag subfamily [3] is characterised by outward recti¢cation ties. To check for speci¢city, Southern blotting was carried out against the RT-PCR products, using a probe spanning the same bases (probe B), prepared by the PCR digoxigenin labelling method with detection as above. *Corresponding author. Fax: (44)-113-3434228. All procedures were carried out according to the manufacturer’s E-mail address: [email protected] (D. Wray). instructions. 0014-5793 / 02 / $22.00 ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies. PII: S0014-5793(02)03055-7 FEBS 26330 17-7-02 M. Ju, D. Wray/FEBS Letters 524 (2002) 204^210 205 2.3. In vitro translation position as in rat eag2 [4,5]. A BLAST search of the human The molecular weight of the heag2 protein was obtained using in genome with the heag2 sequence showed that the correspond- vitro transcription and translation of heag2 cDNA in pGEMHE. For this, heag2 protein was made in the presence of [35S]methionine using ing gene (KCNH5) is located on chromosome 14 at 14q23.1, the Promega T7 TNT Quick coupled reticulocyte lysate system with and the coding sequence comprises 11 exons spanning 338 kb or without canine pancreatic microsomes. Labelled proteins were sep- of genomic sequence. The heag2 cDNA sequence was identi- arated on a 10% SDS^polyacrylamide gel, and exposed to X-ray ¢lm. cal to that of the corresponding exons of the genomic se- 2.4. Electrophysiology quence, except for one base at position 2577 (counting from Xenopus oocytes were injected with 1.5ng or 7 ng RNA respectively the start codon) where the genomic sequence has A instead of for either heag1 or heag2 in 50 nl, and two-electrode voltage-clamp G, without however altering the corresponding amino acid. recordings were made at room temperature (22‡C) as previously de- This may be a polymorphism, although we consistently found W scribed [7]. Oocytes in a 50 l chamber were superfused (1.5ml/min) G in six clones that we sequenced, and also in an EST (ac- with solution containing 2 mM KCl, 115mM NaCl, 10 mM HEPES, cession number U69185). Interestingly, this EST has another 1.8 mM CaCl2, pH 7.2. Cells were held at 380 mV, and leak and capacity subtraction were carried out using 10 mV hyperpolarising base change (G to A at heag2 position 2233), corresponding pulses applied at 0.1 Hz. From the same holding potential, test depo- to a possible mutation/polymorphism A745T in the protein, larisations (500 ms duration) were applied (0.1 Hz) in order to con- although G was present in all but one of the clones sequenced struct current^voltage (I^V) relationships. For heag2, ¢ts to the cur- in the present study. From comparison with ESTs, the start of rent by a Boltzmann function showed deviations, using IV = (V3Er)Gmax/(1+exp((V0:53V)/k)), reversal potential Er = 398.5mV. the polyA signal is apparently located at base 3200. This was apparently because of appreciable heag2 currents at the The heag2 channel protein has a predicted molecular weight 3 3 3 holding potential; instead we ¢tted to IV I380 (I380 I390)(V+80)/ of 112 kDa and isoelectric point of 7.5. In vitro transcription 3 3 10 (currents IV , I380, I390 at voltages V, 80 mV, 90 mV parame- and translation con¢rmed (Fig. 2C) this molecular weight, terised as above) to correct for heag2 current at the holding potential and the subtraction procedure used; good ¢ts were then obtained. where a band of approximately 115kDa was observed. In Student’s t-test was used to test for signi¢cance, and means the presence of microsomes, where N-glycosylation is able to þ S.E.M. are shown. occur, the molecular weight was very similar, implying the absence of marked glycosylation, even though the heag2 se- 3. Results quence displays two consensus sites for N-glycosylation at extracellular locations (Fig. 1). Protein bands were also seen 3.1. Cloning and primary structure at around 105kDa and 65kDa, which might correspond to We obtained a heag2 clone containing an open reading degradation products, or proteins starting from a more down- frame of 2967 bases, corresponding to a protein of 988 amino stream methionine. acids (Fig. 1), taking the start methionine in the homologous Heag2 channel protein is 73% identical to heag1, di¡ering Fig. 1. Sequence alignment of heag1 and heag2 channels. The positions of the characteristic domains are marked. These include the PAS do- main, the core channel transmembrane helices, S1^S6 and the pore P region, the cyclic nucleotide binding region, cNBD, the calcium-dependent calmodulin binding region, CaM, the tetramerisation CAD domain, and a leucine zipper region, LZ.
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