Expression pattern and functional characteristics of two novel splice variants of the two-pore-domain potassium channel TREK-2.
Two novel alternatively spliced isoforms of the human two-pore-domain potassium channel TREK-2 were isolated from cDNA libraries of human kidney and fetal brain. The cDNAs of 2438 base pairs (bp) (TREK-2b) and 2559 bp (TREK-2c) encode proteins of 508 amino acids each. RT-PCR showed that TREK-2b is strongly expressed in kidney (primarily in the proximal tubule) and pancreas, whereas TREK-2c is abundantly expressed in brain. In situ hybridization revealed a very distinct expression pattern of TREK-2c in rat brain which partially overlapped with that of TREK-1. Expression of TREK-2b and TREK-2c in human embryonic kidney (HEK) 293 cells showed that their single-channel characteristics were similar. The slope conductance at negative potentials was 163 +/- 5 pS for TREK-2b and 179 +/- 17 pS for TREK-2c. The mean open and closed times of TREK-2b at -84 mV were 133 +/- 16 and 109 +/- 11 micros, respectively. Application of forskolin decreased the whole-cell current carried by TREK-2b and TREK-2c. The sensitivity to forskolin was abolished by mutating a protein kinase A phosphorylation site at position 364 of TREK-2c (construct S364A). Activation of protein kinase C (PKC) by application of phorbol-12-myristate-13-acetate (PMA) also reduced whole-cell current. However, removal of the putative TREK-2b-specific PKC phosphorylation site (construct T7A) did not affect inhibition by PMA. Our results suggest that alternative splicing of TREK-2 contributes to the diversity of two-pore-domain K+ channels.
Gu
Wenli
W
Institut für Humangenetik, Universität Bonn, Wilhelmstrasse 31, D-53111 Bonn, Germany.
Schlichthörl
Günter
G
Hirsch
Jochen R
JR
Engels
Hartmut
H
Karschin
Christine
C
Karschin
Andreas
A
Derst
Christian
C
Steinlein
Ortrud K
OK
Daut
Jürgen
J
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004594:Electrophysiology; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D024683:Potassium Channels, Tandem Pore Domain; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats / Q000235:genetics / Q000378:metabolism; D014018:Tissue Distribution
D004274:DNA, Recombinant; C503438:KCNK10 protein, human; C503444:Kcnk10 protein, rat; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; D020033:Protein Isoforms; D012333:RNA, Messenger; C103742:potassium channel protein TREK-1