New Ether-à-go-go K+ Channel Family Members Localized in Human Telencephalon

Abstract

A cDNA encoding a novel voltage-gated K+ channel protein was isolated from human brain. This protein, termed BEC1, is 46% identical to rat elk in the ether-à-go-go K+ channel family. The BEC1 gene maps to the 12q13 region of the human genome. Northern blot analysis indicates that BEC1 is exclusively expressed in human brain, where the expression is concentrated in the telencephalic areas such as the cerebral cortex, amygdala, hippocampus, and striatum. By in situ hybridization, BEC1 is detected in the CA1–CA3 pyramidal cell layers and the dentate gyrus granule cell layers of the hippocampus. Specific signals are also found in neocortical neurons. Transfection of mammalian L929 and Chinese hamster ovary cells with BEC1 cDNA induces a voltage-gated outward current with a fast inactivation component. This current is insensitive to tetraethylammonium and quinidine. Additionally, a second related gene BEC2 was isolated from human brain. BEC2 is also brain-specific, located in the neocortex and the striatum, and functional as a channel gene. Phylogenetic analysis indicates that BEC1 and BEC2 constitute a subfamily, together with elk, in the ether-à-go-go family. The two genes may be involved in cellular excitability of restricted neurons in the human central nervous system. A cDNA encoding a novel voltage-gated K+ channel protein was isolated from human brain. This protein, termed BEC1, is 46% identical to rat elk in the ether-à-go-go K+ channel family. The BEC1 gene maps to the 12q13 region of the human genome. Northern blot analysis indicates that BEC1 is exclusively expressed in human brain, where the expression is concentrated in the telencephalic areas such as the cerebral cortex, amygdala, hippocampus, and striatum. By in situ hybridization, BEC1 is detected in the CA1–CA3 pyramidal cell layers and the dentate gyrus granule cell layers of the hippocampus. Specific signals are also found in neocortical neurons. Transfection of mammalian L929 and Chinese hamster ovary cells with BEC1 cDNA induces a voltage-gated outward current with a fast inactivation component. This current is insensitive to tetraethylammonium and quinidine. Additionally, a second related gene BEC2 was isolated from human brain. BEC2 is also brain-specific, located in the neocortex and the striatum, and functional as a channel gene. Phylogenetic analysis indicates that BEC1 and BEC2 constitute a subfamily, together with elk, in the ether-à-go-go family. The two genes may be involved in cellular excitability of restricted neurons in the human central nervous system. ether-à-go-go cyclic nucleotide-binding eag-related gene rapid amplification of cDNA ends expressed sequence tag reverse transcriptase polymerase chain reaction tetraethylammonium base pair(s) kilobases Chinese hamster ovary group of overlapping clones Voltage-gated K+ channels play an essential role in controlling cellular excitability in the nervous system, and regulate a variety of neuronal properties such as interspike membrane potential, action potential waveform, and firing frequency (1Hille B. Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland, MA1992Google Scholar). These results indicate the important functions of voltage-gated K+channels in neuronal signal transduction and processing. Their contribution to behavioral phenotypes such as learning and memory have also been studied (2Ben-Ari Y. Represa A. Trends Neurosci. 1990; 13: 312-318Abstract Full Text PDF PubMed Scopus (308) Google Scholar, 3Engel J.E. Wu C.F. J. Neurosci. 1998; 18: 2254-2267Crossref PubMed Google Scholar). To date, many voltage-gated K+ channel genes have been identified from different tissues such as heart and brain, and constitute an evolutionarily related multigene superfamily (4Strong M. Chandy K.G. Gutman G.A. Mol. Biol. Evol. 1993; 10: 221-242PubMed Google Scholar, 5Deal K.K. England S.K. Tamkun M.M. Pharmacol. Rev. 1996; 76: 49-67Google Scholar, 6Warmke J. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar). This superfamily is classified into two groups, the Shaker and the ether-à-go-go (eag)1 families. The Shaker family is quite heterogeneous and mainly consists of four subfamilies, Kv1–Kv4. Their protein structures are characterized by six transmembrane regions (S1–S6) with a voltage-sensing S4 region and an ion-conducting pore region located between S5 and S6. K+ channels in the eag family also have similar structural features. However, overall sequence similarity between eag and Shaker-type K+ channels is very low. Members of the eag family are related to cyclic nucleotide-gated cation channels, hyperpolarization-activated cation channels and plant hyperpolarization-activated K+ channels, rather than Shaker-type channels (6Warmke J. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar). Indeed, a common feature of the C terminus of eag-type channels is a putative cyclic nucleotide-binding (CNB) domain, a characteristic of such ion channels. The eag family consists of eag, eag-related gene (erg), and elk (6Warmke J. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar, 7Warmke J. Drysdale R. Ganetzky B. Science. 1991; 252: 1560-1562Crossref PubMed Scopus (282) Google Scholar, 8Shi W. Wymore R.S. Wang H.S. Pan Z. Cohen I.S. McKinnon D. Dixon J.E. J. Neurosci. 1997; 17: 9423-9432Crossref PubMed Google Scholar). In Drosophila, genetic mutations of eag or erg induces a hyperexcitable phenotype (9Ganetzky B. Wu C.F. J. Neurogenet. 1983; 1: 17-28Crossref PubMed Scopus (140) Google Scholar, 10Titus S.A. Warmke J.W. Ganetzky B. J. Neurosci. 1997; 17: 875-881Crossref PubMed Google Scholar, 11Wang X. Reynolds E.R. Deak P. Hall L.M. J. Neurosci. 1997; 17: 882-890Crossref PubMed Google Scholar). Human erg maps to LQT2, the locus of inherited long-QT syndrome, an abnormality of cardiac rhythm involving the repolarization of the action potential (12Curran M.E. Splawski I. Timothy K.W. Vincent G.M. Green E.D. Keating M.T. Cell. 1995; 80: 795-803Abstract Full Text PDF PubMed Scopus (2002) Google Scholar). This gene is expressed not only in the heart but also in the brain and parasympathetic ganglia (8Shi W. Wymore R.S. Wang H.S. Pan Z. Cohen I.S. McKinnon D. Dixon J.E. J. Neurosci. 1997; 17: 9423-9432Crossref PubMed Google Scholar). In a dorsal root ganglionic cell line, pharmacological blocking of erg currents causes the disappearance of spike-frequency adaptation of firing (13Chiesa N. Rosati B. Arcangeli A Olivotto M. Wanke E. J. Physiol. 1997; 501: 313-318Crossref PubMed Scopus (150) Google Scholar). This finding suggests that mammalian erg also contributes to the regulation of neuronal excitability. Consequently, we attempted to identify other novel erg-related genes expressed in mammalian brain. As a result, a new gene encoding a voltage-gated K+ channel was isolated, which is more closely related to elk than erg and is exclusively expressed in the human telencephalon. This study reports the molecular cloning, distribution, and channel activity of this gene, BEC1. In addition, the identification of another novel gene closely related to BEC1 is described, indicating the existence of a new subfamily in the eag K+ channel family. A BLAST search of the expressed sequence tag (EST) data base of GenBank, using human erg as a query amino acid sequence, retrieved two sequences with the accession number R35526 (387 bases) and M79045 (231 bases). To identify the 5′ and 3′ ends of the cDNA corresponding to each EST, 5′ and 3′ rapid amplification of cDNA ends (RACE) were performed using the Human Brain Marathon-Ready cDNA (CLONTECH) and primers derived from each EST sequence. Amplified fragments were directly cloned into the plasmid pCR2.1 (Invitrogen). Sequencing the RACE products revealed that the two EST were part of a cDNA. Determined sequences were assembled into a large contig (3610 bases) with an open-reading frame encoding 1083 amino acids, which we called human BEC1. Finally, to verify that human BEC1 cDNA including the entire open-reading frame could be cloned from an independent source, human poly(A)+ RNA (CLONTECH) was used for reverse transcriptase-polymerase chain reaction (RT-PCR). Reverse transcriptase reaction was primed with the random hexamer. Primers for PCR were designed from the 3610-bp contig as follows: 5′-GGAATTCC CTA AGA TGC CGG CCA TGC-3′ and 5′-GCTCTAGAGC ACT CTG AGG TTG GGC CGA AC-3′, which contains artificial sequences with Eco RI and Xba I sites, respectively (italic). Cycles were as follows: initial denature at 96 °C for 1 min; and 35 cycles of denaturation at 96 °C for 10 s, annealing at 68 °C for 30 s, and extension at 72 °C for 7 min. Pfu DNA polymerase (Stratagene) was used. The RT-PCR exclusively generated a 3.3-kb fragment. The amplified fragment was digested with Eco RI and Xba I, and cloned into pME18S, a plasmid for mammalian expression (14Maruyama K. Takebe Y. Med. Immunol. 1990; 20: 27-32Google Scholar). The resulting BEC1 expression vector was termed pME-E1. To isolate rat homolog cDNAs of BEC1, RT-PCR was performed using primers derived from two nucleotide sequences of BEC1 conserved with elk, 5′-ACc TTC CTG GAC ACC ATC GC-3′ and 5′-CCa AAc ACC ACc GCg TGC AT-3′, in which lowercase letters are indicated mismatches with elk. These sequences correspond to amino acid residues 13–19 (TFLDTIA) and 492–498 (MHAVVFG) of human BEC1, which are shared only with elk among the eag family (Fig. 1). Rat brain poly(A)+ RNA was prepared by guanidine thiocyanate extraction followed by oligo(dT)-cellulose chromatography (15Sugimoto T. Saito M. Mochizuki M. Tahara A. Watanabe U. Hashimoto S. Kawashima H. Saito T. Kurokawa K. Yoshida S. Neurohypophysis: Recent Progress of Vasopressin and Oxytocin Research. Elsevier Science Publishers B.V., Amsterdam1995: 409-413Google Scholar), and converted into random-primed cDNA. The RT-PCR amplified two fragments of 1.5 and 1.4 kb, which encode polypeptides with 97% and 59% identities to human BEC1, respectively. Because the N and C termini were missing, 5′- and 3′-RACE were performed on the basis of each determined sequence using the Rat Brain Marathon-Ready cDNA (CLONTECH). Sequences of the 1.5-kb fragment and its 5′- and 3′-RACE products were assembled into a contig (3715 bases) with an open reading frame of 1087 amino acids, while sequences of the 1.4-kb fragment and its RACE were merged a contig (3736 bases) with an open reading frame of 1017 amino acids. The 1087-amino acid sequence is extremely similar (95%) to human BEC1, indicating that this is the rat counterpart of BEC1. Another sequence shares 48% sequence similarity with BEC1. We estimated that the latter gene was a homolog to BEC1, which we named BEC2. BEC1 sequences are markedly conserved between the human and the rat. Nucleotide identity is 89% between both open reading frames of human and rat BEC1. If human BEC2 cDNA is also extremely similar to the rat one, human BEC2 cDNA will be amplified by RT-PCR using some primer sets derived from rat sequence. Thus, to identify human BEC2, RT-PCR with human poly(A)+RNA was performed using primers designed from the sequences around the initiation and of rat BEC2 and AGG A fragment was amplified by the RT-PCR cycles of and 68 with directly cloned into pCR2.1 and The sequence an open reading frame encoding 1017 amino with 89% identity to rat BEC2, that this cDNA human BEC2. 5′- and ends were determined by The results revealed a contig bases) of human BEC2 cDNA and the of only a in the reverse To an expression vector for human BEC2, the open reading frame of human BEC2 cDNA in pCR2.1 was into This vector was named of each fragment were using DNA and an DNA sequence and analysis were performed using the 1994; PubMed Scopus Google Scholar), and of the N. M. Mol. Biol. Evol. Google Scholar), respectively. Northern blot analysis of was performed using Northern on which of poly(A)+ been BEC1 was determined using a corresponding to amino of BEC1. The were at in and and for 30 in at The were to with two at °C for To BEC2 distribution, the were as with a corresponding to amino of BEC2, for 30 in at and to for 10 of human erg was determined as a gene using a corresponding to amino RT-PCR analysis of human BEC2, the primers GGC and GC-3′ were used. This primer a fragment corresponding to amino of BEC2. cDNAs were from each human poly(A)+ RNA of each cDNA was amplified by cycles of PCR 72 1 with of amplified products were by on a and by The plasmid BEC1 or BEC2 cDNA was used as a The of cDNA was estimated by RT-PCR of the cDNA A. Mochizuki S. Y. S. Mol. Pharmacol. 1995; Google Scholar). In situ was performed as K. Y. K. H. H. T. K. Yoshida T. J. 1997; PubMed Scopus Google using rat brain and a RNA corresponding to amino of rat BEC1. The of were in in and at 7 hybridization, the were in for with in with and The RNA was with RNA polymerase using a RNA from a fragment of BEC1 cDNA with a sequence by was performed at °C with the RNA in and E. hybridization, were with A at °C for 30 and at °C with and for The was detected with a acid with the was performed identical as a L929 and cells were with BEC1 cDNA for as S. A. K. Google Scholar, M. T. K. Y. Y. H. J. Y. M. T. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). L929 cells were with the BEC1 expression vector and the protein expression vector (CLONTECH) using the cells were determined by protein with an BEC1 cDNA was into a the gene of the mammalian expression plasmid S. S. 1990; 18: PubMed Scopus Google Scholar). cells in gene were with this plasmid using and in BEC1 expression was amplified with at of BEC2, L929 cells were with and as were performed from BEC1 using in S. A. K. Google Scholar). were using of The 10 The 10 were at and were from erg-related the accession R35526 were identified in the EST data base of using a BLAST search with an amino acid sequence of human erg (6Warmke J. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar). R35526 (387 bases) two regions encoding amino acid sequences with and identities to and acid around the and regions of human respectively M79045 (231 bases) a sequence with identity to a acid of the S5 region As a of RACE using primers derived from each EST sequences to identification a cDNA (3610 bases) with an open-reading frame encoding 1083 amino identity to K+ channels of the eag family. The similarity is to rat elk, which been identified only W. Wang H.S. Pan Z. Wymore R.S. Cohen I.S. McKinnon D. Dixon J. J. Physiol. 1998; PubMed Scopus Google Scholar). Rat eag and human erg and sequence similarity with BEC1, respectively. We named this new protein BEC1. Additionally, rat counterpart of the BEC1 gene was identified in rat brain which 1087 amino with identity to human BEC1. Another related gene, termed BEC2, was identified in rat or human brain human and rat BEC2 of 1017 amino with 48% identity to BEC1. acid sequences of BEC2 are conserved between human and rat the part of BEC2 with amino is rather with BEC1. The sequence of BEC1 and BEC2 is in BEC1 and BEC2 a corresponding to the six transmembrane and the pore region of voltage-gated K+ channels. The is conserved between BEC1 and BEC2 with This sequence similarity is to that among in a subfamily of the K+ channel superfamily The putative voltage-sensing S4 and ion-conducting pore regions are also shared by BEC1 and BEC2. In the S4 only amino are different between BEC1 and BEC2. The S4 regions of both BEC1 and BEC2 residues found at Additionally, other two and residues are found at common The pore regions of BEC1 and BEC2 have a a common of eag-type K+ channels (6Warmke J. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar), and four amino from each were found in the between the S5 and pore BEC1 contains and BEC2 was identified between the and S4 region of BEC2. In the is a sequence with similarity to the of cyclic nucleotide-binding such as cyclic nucleotide-gated cation channels and hyperpolarization-activated cation channels. the of BEC1 and BEC2 are to eag-type K+ channels, are markedly similar to each other among the cyclic nucleotide-binding These results indicate that BEC1 and BEC2 may be of a new subfamily of eag-type analysis suggests that novel genes have a common with eag-type channels and an in the eag family Human EST which to BEC1, is a sequence of the in the human brain cDNA by P. A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). This been termed and to the 12q13 region of the human using in situ analysis by S. E. A. A. S. E. M. A. 1996; 13: PubMed Scopus Google Scholar). The sequence of was to number and consists of a sequence of the used in the We performed sequence of human BEC1 and The revealed that the sequence of with was identical to the sequence of human BEC1 of a in 10 to of BEC1. region been identified by in situ with indicating that BEC1 is derived from an identical gene to the BEC1 locus is located in the 12q13 Northern blot analysis revealed the of a BEC1 in human brain poly(A)+ RNA The of the with the of identified cDNA. signals were detected in other human tissues including the and human brain, of BEC1 were detected in structures such as the cerebral cortex, amygdala, and hippocampus, and the regions including the and (Fig. In brain an was but of only the was found in human but the expression was very low. BEC1 were not in the or in the which contains and Thus, the expression of the BEC1 is restricted to the in human The expression of BEC1 with that of human which a more in the brain (Fig. of BEC2 was also by Northern blot analysis of human poly(A)+ signals were detected only in the brain, with and We have identified BEC2 cDNA with as an of a the to the identified cDNA. the signals were of BEC2 was determined by RT-PCR A fragment corresponding to BEC2 was amplified only from the brain This fragment was generated the plasmid BEC2 cDNA was used as a but not BEC1 cDNA. These results suggests that BEC2 is exclusively expressed in the brain, as as BEC1. Northern blot analysis of the brain regions indicated the BEC2 expression restricted to the similar to BEC1 (Fig. BEC2 were detected in the regions such as the and In addition, signals for BEC2 were detected in the cerebral and hippocampus. The expression of BEC2 is to that of BEC1, the expression to be different in each In situ was performed using rat brain to the cellular of BEC1 the brain. signals were found in the hippocampus, only the to BEC1 was used (Fig. A and Specific signals were also in the cerebral In the hippocampus, in situ that BEC1 were concentrated in the pyramidal cell layers of the and and in the granule cell layers of the dentate In the cerebral cortex, BEC1 signals were from to Specific signals were detected in cell of neurons with pyramidal in the cerebral (Fig. C and To properties of BEC1 using the L929 cells were with the BEC1 expression The cells were at a potential of and were to between and an outward current in cells the was to the outward current was and to a The current by to was were not in cells (Fig. for the current and the current at the of the which correspond to and respectively. The current from to In the current to and with current analysis of BEC1, were to from a potential of and were to between and (Fig. The BEC1 current was and by the repolarization from inactivation and a The current at in the the potential for this that BEC1 is a of the K+ channel family. We the of two K+ channel and on the BEC1 was used as a K+ current of the channel but not that of the BEC1 channel in to not In K+ channels of the eag family are to with the Shaker family T. 1996; PubMed Scopus Google Scholar, J. H. A. A. W. J. 1994; 13: PubMed Scopus Google Scholar). results indicate that BEC1 also the common to are heterogeneous in the eag family. the erg current with an of T. 1997; 80: PubMed Scopus Google Scholar), the of eag current is J. H. A. A. W. J. 1994; 13: PubMed Scopus Google Scholar). BEC1 was insensitive to 10 which the channel activity of not BEC1 were using cells and the gene amplification by The was as a of BEC1. This cell an outward current in to not as L929 cells with BEC1 cDNA. The cells These results that the identified current is by BEC1. The of L929 cells and the were used to identify channel activity of BEC2, as as BEC1. As in an outward current in indicating that the BEC2 gene also a channel The inactivation such as the BEC1 current was not in the BEC2 This study new of the voltage-gated K+ channel BEC1 and BEC2, which are exclusively expressed in human brain. a number of voltage-gated K+ channel genes have been isolated from the mammalian brain, of genes are also expressed in other tissues such as the heart and the Shaker-type channel genes identified in the brain, and are in heart or J.E. McKinnon D. 1994; PubMed Scopus Google Scholar, P. E. B. J. 1996; PubMed Scopus Google Scholar, S. M. M. B. Gutman G.A. Chandy K.G. PubMed Scopus Google Scholar). and are expressed in B. E. A. P. J. M. J. Biol. Full Text PDF PubMed Google Scholar, S. S. B. Gutman G.A. Chandy K. J. Biol. Full Text PDF PubMed Google Scholar). of mammalian erg also is in heart M.E. Splawski I. Timothy K.W. Vincent G.M. Green E.D. Keating M.T. Cell. 1995; 80: 795-803Abstract Full Text PDF PubMed Scopus (2002) Google expression in the brain is a feature of BEC1 and BEC2. In addition, both are concentrated in the of the human brain, that this channel may to excitability of restricted neurons in the central nervous system. BEC1 in the are detected in the and pyramidal neurons and the dentate gyrus granule which constitute the a important to and The and Scholar). the contribution of voltage-gated K+ channels to learning and is of to study of BEC1 channel in this BEC1 to the eag family of voltage-gated K+ channels. of BEC2, a gene closely related to BEC1, the existence of a new subfamily of eag-type channels. BEC1 and BEC2 are to elk, a putative K+ channel gene. The identified rat homolog of elk, rat W. Wang H.S. Pan Z. Wymore R.S. Cohen I.S. McKinnon D. Dixon J. J. Physiol. 1998; PubMed Scopus Google Scholar), is not identical but is the closely related to BEC1 and BEC2. we have isolated a cDNA of a human gene, and S. Rat the rat of this gene is identical to Thus, this subfamily consists of at mammalian of elk. BEC1 an outward current with a fast inactivation component. currents are also by Shaker-type channels such as and K.K. England S.K. Tamkun M.M. Pharmacol. Rev. 1996; 76: 49-67Google Scholar). However, Shaker-type channels, the BEC1 current not only the but also a the The between of both is a feature of the BEC1 In BEC2 a outward similarity between BEC1 and BEC2 is in the region important to channel two sequences may be to identify amino the between channel mutations in K+ channel genes have been identified as genes for inherited (12Curran M.E. Splawski I. Timothy K.W. Vincent G.M. Green E.D. Keating M.T. Cell. 1995; 80: 795-803Abstract Full Text PDF PubMed Scopus (2002) Google Scholar, E.R. P. M. 1994; PubMed Scopus Google Scholar, M.E. Splawski I. J. Timothy K.W. Vincent G.M. G.M. Keating M.T. 1996; PubMed Scopus Google Scholar, P. Science. 1998; PubMed Scopus Google Scholar). Thus, the that BEC1 with be The BEC1 gene maps to the 12q13 region of the human genome. To date, genetic have been to the 12q13 for a of M.M. 1997; PubMed Google and H. K. M. A. B. T. U. M. N. J. Med. 1997; PubMed Scopus Google Scholar). using the BEC1 gene are to BEC1 is to such The nucleotide at of 10 of BEC1 is by in as This is a nucleotide BEC1. In the identification of BEC1 and BEC2 genes to the of the K+ channel multigene We have that both genes are in human and encode channel may new into neuronal signal transduction and in human telencephalon. We and for and we and for We also E. for this