Expression pattern and functional characteristics of two novel splice variants of the two-pore-domain potassium channel TREK-2

Abstract

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.

Figure 1. Sequence analysis of TREK-2 splice forms

A, relative orientation of the first exons of the splice variant identified in kidney (TREK-2b), fetal brain (TREK-2c) and the variant reported by Lesage et al. 2000b (TREK-2a). The corresponding genomic contigs are also shown. Exons are not drawn to scale. B, N-terminal cDNA and amino acid sequences of the human TREK-2 splice variants (accession numbers are given in brackets). The start codon (bold) and the splice sites of the first intron (underlined) are indicated. cDNA sequences are shown in capital letters, intronic sequences in lower case letters.

Figure 2. Tissue distribution of human TREK-2b and TREK-2c

Multiple-tissue cDNA panels from Clontech were used to study the tissue distribution of the three TREK-2 isoforms. To avoid possible genomic contamination, we used splice variant-specific primer pairs that included an exon-intron boundary fragment. In panel Human I, 180 bp fragments were amplified by specific TREK-2b and TREK-2c primers, whereas no PCR product could be observed with specific TREK-2a primers (not shown). Note that the TREK-2b signals in lung, liver and muscle were very faint. All cDNAs were also tested for GAPDH expression, and negative controls were performed with all RNA samples to exclude contamination.

Figure 3. Expression of TREK-2b in human kidney

RT-PCR analysis of human glomeruli (GLO), proximal tubule (PT), thick ascending limb (TAL) and cortical collecting duct (CD). Molecular weight markers are shown in the left lane.

Figure 4. Distribution of TREK-2c and TREK-1 transcripts in rat brain

A-C, TREK-2c; D-F, TREK-1. X-ray film images show the differential expression patterns in adjacent midsagittal sections (A and D), and in coronal forebrain (B and E) and brainstem (C and F) sections. AM, anteromedial thalamic n.; AP, area postrema; Arc, arcuate n.; CA2 and CA3, hippocampal pyramidal cells of the corresponding region; Cb gr, cerebellar granule cell layer; CPu, caudate putamen; DG, hippocampus dentate gyrus; DTg, dorsal tegmental n.; Hy, hypothalamus; IP, interpeduncular n.; LC, locus coeruleus; LOT, n. of the lateral olfactory tract; Ly IV, neocortical layer IV; Mo5, motor trigeminal n.; MPO, medial preoptic n.; MVPO, medioventral periolivary n.; OB gr, olfactory bulb granule cell layer; Pir, piriform cortex; Pn, pontine n.; PVA, paraventricular thalamic n.; SO, supraoptic n.; Sol, solitary n., where n. stands for nucleus; scale bars represent 5 mm.

Figure 5. Single-channel recordings of TREK-2c

A, typical cell-attached recording of a TREK-2c channel expressed in HEK 293 cells. The top trace shows the first 20 ms of the lower trace at 50 × higher time resolution. B, all-points amplitude histogram of a typical cell attached record form a patch containing only one TREK-2c channel. C, event amplitude histogram constructed from the same set of data using an algorithm that excludes very short events, i.e. only events with a duration of more than 0.3 ms (> 5 data points) were included. D, typical open time distribution. E, closed time distribution of the same recording. In A-E the transmembrane potential (inside-outside) was −84 mV, the sampling rate was 16 kHz, and the pipette solution contained no divalent cations.

Figure 6. Dependence of single-channel conductance on external Ca²⁺

A, single-channel current-voltage relation of TREK-2b and TREK-2c in the presence (•) and in the absence (○) of external divalent cations. B, mean values of the slope conductance of TREK-2b and TREK-2c, determined between −80 and −40 mV in the presence (•) and in the absence (○) of external divalent cations. C, the slope conductance of TREK-2b and TREK-2c measured in the presence (+DVC) and in the absence (-DVC) of external divalent cations.

Figure 7. Whole-cell currents through TREK-2b and TREK-2c channels

Typical whole-cell currents in physiological salt solution (containing 5 mm K⁺) observed after transfection of HEK 293 cells with TREK-2b or TREK-2c.

Figure 8. Effects of protein kinase C activation

A, typical recording of the effects of 40 nm PMA on the whole-cell current carried by TREK-2c; repetitive voltage ramps between −120 and +40 mV were applied. B, comparison of the current-voltage relation obtained under control conditions and in the steady state after application of PMA. Inset, comparison of the effects of 40 nm PMA on the currents produced by TREK-2b, TREK-2c and TREK-2c T7A.

Figure 9. Effects of protein kinase A activation

A, typical recording of the effects of 10 μm forskolin on the whole-cell current carried by TREK-2c; repetitive voltage ramps between −120 and +40 mV were applied. B, comparison of the current-voltage relation obtained under control conditions and in the steady state after application of forskolin. Inset, comparison of the effects of 10 μm forskolin on the currents produced by TREK-2b, TREK-2c and TREK-2c S353A.