Research reportCloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel
Introduction
Potassium channels occur in all mammalian cell types, performing functions in both excitable and non-excitable cells. More than 50 potassium channels have now been identified in humans and these can be divided into distinct families based on both primary sequence features and pharmacology. The potassium (K+) channels are characterised by having 2, 4 or 6 transmembrane regions and 1 or 2 pore domains. The most recent class of K+ channels to be identified has 4 transmembrane domains and 2 pore domains, the 2P/4TM K+ channel family, also called the tandem pore domain potassium channel family. The first member of the family is known as TWIK-1 [8] (tandem of P domains in a weak inward rectifying K+ channel). These tandem pore domains retain a tripeptide sequence motif G(Y/F/L)G present in all K+ channels which is reported to act as the selectivity filter of the pore [5]. An exception to this observation is the recently reported KCNK7 [20], this has the unusual tripeptide GLE in the second pore domain, although importantly no channel activity has yet been demonstrated for this gene. TWIK-1, the initial gene in the family, was demonstrated to function as a weakly inward rectifying K+ channel [8]. Subsequent members of the family have been given names which both relate them to TWIK-1 and broadly indicate their properties: thus TWIK-1 and 2 [2] are weakly inwardly rectifying K+ channels, TASK-1 [3], 2 [17] and 3 [7] are acid-sensitive K+ channels and TREK-1 [12] and TRAAK (arachidonic acid stimulated) [11] are outwardly rectifying mechanosensitive K+ channels.
There are currently six reported functional human 2P domain K+ channels each with its own tissue distribution pattern. TWIK-1 is widely distributed, being particularly abundant in the brain and heart [8]; TWIK-2 is also widely expressed [2]; TREK-1 has its highest expression levels broadly distributed in the CNS [12]; TRAAK has its highest expression levels in brain and placenta [11]; TASK-1 is expressed in many tissues most significantly pancreas and placenta [3]; TASK-2 is mainly expressed in the kidney and largely absent from the brain [17]. The 2P/4TM K+ channel family is extended to seven functional members by the recently reported, rat TASK-3, this is expressed in many rat tissues including brain, kidney, liver, lung, colon, stomach, spleen, testis and skeletal muscle [7].
The Caenorhabditis elegans genome is reported to code for over 80 potassium channel proteins of which 50 are 2P/4TM family members [18]. In the case of Drosophila melanogaster, there are 30 K+ channels of which 11 are 2P/4TM K+ channel family members [18].
This paper reports the identification, cloning and functional expression of a novel human 2P/4TM K+ channel. This is the 7th human 2P/4TM K+ channel family member which has been demonstrated to be functional. It shares greatest amino acid sequence homology (62% identity) with TASK-1, an acid-sensitive 2P domain K+ channel. The gene sequence reported here shows 75% nucleotide identity to the very recently reported rat TASK-3 gene [7], indeed we consider this gene to be the human orthologue, hTASK-3. There is, however, significant sequence divergence between the 3′ ends of the human and rat TASK-3 genes, after the 4th TM domain. This is discussed further.
Section snippets
Gene cloning — degenerate PCR
Known members (human, mouse and rat) of the 2P domain potassium channel family were aligned at both the protein and the DNA levels (Pile-up, GCG). The most significant conservation of sequence is found in the two pore domains (P1 and P2) and in transmembrane domain 2 (TM2). Degenerate PCR primers were designed to amplify specific fragments between these highly conserved regions of the known members of the 2P domain potassium channel family. A second, nested, PCR, with a second set of degenerate
Cloning/primary structure
The degenerate PCR products were subcloned and 48 clones were DNA sequenced: of these 26 clones analysed had a 99% match to TASK-1 (KCNK3, Genbank AF006823) and 10 clones formed a contig with 98% match to TWIK-1 (KCNK1, Genbank U90065). 3 clones formed a 352 bp contig which had an 80% match to TASK-1 in the P1 to P2 region, this novel sequence was extended in both the 5′ and 3′ directions by RACE PCR to obtain sequence data for the full-length gene. The component sections of sequence data were
Discussion
The human gene cloned in this paper represents a novel member of the growing family of 2P/4TM K+ channels. We have compared the primary sequence of the gene (374 aa) reported in this paper with the recently identified [7] rat TASK-3 sequence (395 aa). The sequence identity to the rTASK-3 at the amino acid level is 74%: we therefore believe our novel gene to be the human orthologue (hTASK-3) of rTASK-3. This level of homology is rather lower than that seen for examples of other, mammalian,
Acknowledgements
Human brain tissue was obtained from the Netherlands Brain Bank in Amsterdam (Co-ordinator Dr R. Ravid).
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