Functional properties of four splice variants of a human pancreatic tandem-pore K+ channel, TALK-1

Jaehee Han,1,2 Dawon Kang,2 and Donghee Kim2

2Department of Physiology and Biophysics, Finch University of Health Sciences/Chicago Medical School, North Chicago, Illinois 60064; and 1Department of Physiology, Gyeongsang National University School of Medicine, Chinju 660-751, Korea

Submitted 26 December 2002 ; accepted in final form 22 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TALK-1a, originally isolated from human pancreas, is a member of the tandem-pore K+ channel family. We identified and characterized three novel splice variants of TALK-1 from human pancreas. The cDNAs of TALK-1b, TALK-1c, and TALK-1d encode putative proteins of 294, 322, and 262 amino acids, respectively. TALK-1a and TALK-1b possessed all four transmembrane segments, whereas TALK-1c and TALK-1d lacked the fourth transmembrane domain because of deletion of exon 5. Northern blot analysis showed that among the 15 tissues examined, TALK-1 was expressed mainly in the pancreas. TALK-1a and TALK-1b, but not TALK-1c and TALK-1d, could be functionally expressed in COS-7 cells. Like TALK-1a, TALK-1b was a K+-selective channel that was active at rest. Single-channel openings of TALK-1a and TALK-1b were extremely brief such that the mean open time was <0.2 ms. In symmetrical 150 mM KCl, the apparent single-channel conductances of TALK-1a and TALK-1b were 23 ± 3 and 21 ± 2 pS at –60 mV and 11 ± 2 and 10 ± 2 pS at +60 mV, respectively. TALK-1b whole cell current was inhibited 31% by 1 mM Ba2+ and 71% by 1 mM quinidine but was not affected by 1 mM tetraethylammonium, 1 mM Cs+, and 100 µM 4-aminopyridine. Similar to TALK-1a, TALK-1b was sensitive to changes in external pH. Acid conditions inhibited and alkaline conditions activated TALK-1a and TALK-1b, with a K1/2 at pH 7.16 and 7.21, respectively. These results indicate that at least two functional TALK-1 variants are present and may serve as background K+ currents in certain cells of the human pancreas.

tandem-pore potassium channel; background potassium channel; pancreas; pH


THE MEMBERS of the tandem-pore K+ (K2P) channel family are defined by their distinct structural features, which are different from those of the voltage-gated, ATP-sensitive, and G protein-gated K+ channel families. An individual subunit of a K2P channel possesses four transmembrane segments and two pore-forming domains, and a functional channel is believed to be a dimer (13, 25). At present, there are 14 members within the K2P family and they can be divided into five subfamilies based on phylogenetic analysis. These include TWIK (5, 24, 31, 32), THIK (33), TASK (9, 18, 21), TALK (8, 12), and TREK/TRAAK subfamilies (2, 10, 11). Some of the K2P channels form functional K+ channels when expressed in oocytes or mammalian cell lines (COS, HEK), whereas others (THIK-2 and TASK-5) do not. Those that form functional channels are usually active across the physiological range of membrane potentials and show no time-dependent inactivation, consistent with their role as resting or background K+ channels. In addition, certain K2P channels are modulated by a variety of signaling molecules that include receptor agonists, by changes in extracellular (pHo) and intracellular pH (pHi), and by cell swelling/membrane stretch and free fatty acids (16, 28). Therefore, K2P channels are likely to serve as key determinants of cell excitability and also provide a modulatory influence on cell function as targets of different physiological changes.

Studies over the past 5 years have revealed the electrophysiological and pharmacological properties of most of the functional K2P channels. Native K+ channels that might be functional correlates of several, but not all, cloned K2P channels have also been identified in various cell types. Thus, native K+ channels with properties similar to TASK-1, TASK-3, TREK-1, and TREK-2 were recently reported in various regions of the brain and in other tissues (4, 7, 15, 17, 19, 22, 37). The identification of native K+ channels that are functional correlates of cloned K2P channels has been possible because of the detailed knowledge of their single-channel kinetics and sensitivity to various pharmacological agents. A few of the K2P channels such as TALK and THIK, however, have yet to be characterized at the single-channel level to help identify their native counterparts.

The diversity of functional K2P channels can be increased by heteromultimerization among different K2P channel subunits. For example, coexpression of TASK-1 and TASK-3 in oocytes enabled the formation of a functional heteromeric channel (6, 36). So far, there is no conclusive evidence showing that any native K2P channel is a heteromer. Another way that nature has increased the number of functional K2P channels is through alternative splicing. Two splice variants of TASK-1, two variants of TREK-1, and three variants of human TREK-2 have been identified (3, 10, 14, 22). Therefore, it is likely that splice variants of other K2P channels also exist. The detailed characterization of single-channel properties of all splice variants is necessary to help identify the native K2P channel forms in different tissues and to study their physiological roles.

The aim of this study was therefore to identify potential splice variants of TALK-1, determine which variants are functional, and characterize in detail the single-channel kinetics of all functional variants. Because TALK-1a originally cloned from human pancreas is activated by alkaline conditions, we also tested whether any of the splice variants is also sensitive to changes in pHo. Here we report isolation of three novel splice variants of TALK-1 from human pancreas and single-channel characterization of two functional variants, TALK-1a and TALK-1b. Our results show that both TALK-1a and TALK-1b are highly sensitive to pHo near the physiological range.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nucleotide sequences reported in this paper have been submitted to GenBank with accession numbers AY253145 (TALK-1b), AY253146 (TALK-1c), and AY253147 (TALK-1d).

Cloning of cDNAs. Human pancreas total RNA (Clontech, Palo Alto, CA) was reverse-transcribed to prepare first-strand cDNA with the Superscript preamplification system (Life Technologies, Rockville, MD). Specific primers [5'-ATA TGC CCA GTG CTG GGC TCT GCA GCT G-3' (sense) and 5'-TCA GCT TCC CAG TCC TTT CTT GGA TAT GGG GA-3' (antisense)] for TALK-1a were used in a polymerase chain reaction (PCR) with ExTaq polymerase. PCR conditions were 94°C for 3 min, 30 cycles of 94°C (40 s)-50°C (60 s)-72°C (2 min), and finally 72°C for 10 min. Amplified products (~0.8 to ~1.2 kb) were subcloned into pcDNA3.1 TOPO vector (Invitrogen, Carlsbad, CA) by TA cloning. To isolate another potential TALK-1 variant homologous to the mouse TALK-1, two specific primers [5'-ATA TGC CCA GTG CTG GGC TCT GCA GCT G-3' (sense) and 5'-TCA TGC AGA GAT GGG GAT TTT CTG TG-3' (antisense)] were used in a PCR with human pancreas cDNA as template. An amplified DNA product (~0.9 kb) was detected and subcloned into the pcDNA3.1 TOPO vector by TA cloning. Plasmid DNA was isolated from three separate colonies from each cloning procedure, and the subcloned DNA fragments were sequenced on both strands with the dideoxy termination method.

To confirm the expression of TALK-1 variants in human pancreas, additional reverse transcriptase (RT)-PCRs were carried out with specific primers designed from exons 4, 5, and 6, where the splicing occurred. The primers used were from exons 4 and 6 (5-'GGGACGCTGGTCATTCTCATCTTCC-3' and 5'-TTGGACTCCTCTTGCTGCTGTAGAGCC-3') for TALK-1a and -1d, from exon 5' (5'-GGGACGCTGGTCATTCTCATCTTCC-3' and 5'-CGCTCATGCAGAGATGGGGATTTTCT-3') for TALK-1b, and from exon 6' (5'-CCACCCCCTTAACTTCATCACTCCCTC-3' and 5'-TTGGACTCCTCTTGCTGCTGTAGAGCC-3') for TALK-1c. The same amount of cDNA was used for all reactions. All PCR conditions were 94°C for 3 min, 30 cycles of 94°C (30 s)-50°C (20 s)-72°C (1 min), and finally 72°C for 10 min. A densitometer was used to assess the relative expression of TALK-1a and TALK-1d. DNA bands were extracted from the gel, amplified again with the same primers, and sequenced to confirm the presence of each variant. These results were reproduced in three different cDNA preparations.

Northern blot analysis. Multiple human tissue Northern blots consisting of 15 different tissues were purchased from OriGene Technologies (Rockville, MD). Membranes were pre-hybridized for 30 min at 65°C and hybridized for 1 h at 65°C in ExpressHyb solution (Clontech) with 32P-labeled TALK-1b cDNA following the manufacturer's protocol. The membrane were rinsed with solution containing 2x SSC (3.0 M sodium chloride and 0.3 M sodium citrate) and 0.05% sodium dodecyl sulfate (SDS) for 30 min at room temperature. A second washing was performed in solution containing 0.1x SSC and 0.1% SDS for 40 min at 50°C. The membrane was exposed to X-ray film and developed 50 h later. The membrane was probed again with 32P-labeled {beta}-actin DNA.

Transfection in COS-7 cells. Human (h)TALK-1 variants subcloned into pcDNA3.1 vector were tested for functional expression in COS-7 cells. COS-7 cells were seeded at a density of 2 x 105 cells per 35-mm dish 24 h before transfection in 10% bovine serum in Dulbecco's modified Eagle's medium (DMEM). COS-7 cells were cotransfected with pcDNA3.1/TALK-1 and pcDNA3.1/green fluorescent protein (GFP) with LipofectAMINE and OPTI-MEM I reduced-serum medium (Life Technologies). Green fluorescence from cells expressing GFP was detected with the aid of a Nikon microscope equipped with a mercury lamp light source. Cells were used 2–3 days after transfection.

Electrophysiological studies. Electrophysiological recording was performed with a patch-clamp amplifier (Axopatch 200; Axon Instruments, Union City, CA). All recordings were performed at room temperature (24°C). Single-channel currents were digitized with a digital data recorder (VR10, Instrutech, Great Neck, NY) and stored on videotape. The recorded signal was filtered at 5 kHz with an eight-pole Bessel filter (–3 dB; Frequency Devices, Haverhill, MA) and transferred to a computer (Dell) with the Digidata 1200 interface (Axon Instruments) at a sampling rate of 20 kHz. Threshold detection of channel openings was set at 50%. Single-channel data were analyzed to obtain duration histogram, amplitude histogram, and relative channel activity (relative NPo, where N is the number of channels in the patch, and Po is the probability of a channel being open) with the pCLAMP program (version 7). NPo was determined from ~1 min of current recording. Single-channel current tracings shown in Figs. 6 and 7 were filtered at 2 kHz. In experiments using excised patches, pipette and bath solutions contained (in mM) 150 KCl, 1 MgCl2, 5 EGTA, and 10 HEPES (pH 7.3). For whole-cell recordings, bath solution contained (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.3). Arachidonic acid was dissolved by sonicating for 5 min (Heat Systems-Ultrasonics W-380, Farmingdale, NY) in bath recording solution at the desired concentration. All other chemicals were purchased from Sigma (St. Louis, MO). For statistics, analysis of variance and Student's t-test were used, with P < 0.05 as a criterion for significance. Data are presented as means ± SD.



View larger version (57K):
[in this window]
[in a new window]
 
Fig. 6. Single-channel properties of TALK-1a and TALK-1b. A: cell-attached patches were formed from COS-7 cells expressing TALK-1a, and single-channel openings were recorded at various membrane potentials are shown. Dotted lines indicate the open state and were drawn by eye. B: open-time ({tau}o) and amplitude histograms of TALK-1a were obtained from openings at –60 and +60 mV. C: cell-attached patches were formed from COS-7 cells expressing TALK-1b, and single-channel openings recorded at various membrane potentials are shown. D: open-time and amplitude histograms of TALK-1b were obtained from openings at –60 and +60 mV. E: current amplitudes from the first open level were determined from amplitude histograms at each membrane potential to draw the current-voltage relationship. Each point is the mean ± SD of 3 determinations.

 


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 7. pH-dependent changes in hTALK-1b current in COS-7 cells. A: whole cell currents were recorded after ramp pulses (–120 to + 60 mV, 1-s duration) to generate current-voltage relations at different pH values. B: relative currents at +60 mV of TALK-1a and TALK-1b were determined and plotted as a function of extracellular pH (pHo). Points were fitted to a Boltzmann equation (see text). pH values at which the half-maximal effect occurred were 7.2 for both variants of TALK-1. Each point is the mean ± SD of 5 determinations. C: single-channel openings from an outside-out patch at different pHo values. Internal pH (pHi) was kept at 7.3. Amplitude histograms at each pHo are also shown. D: graph shows effect of changes in pH on TALK-1b current calculated from experiments using outside-out and inside-out patches. Inside-out patches were used to study the effect of changes in pHi. Each point is the mean ± SD of 5 determinations. *Significant difference from corresponding control value obtained at pH 7.3 (P < 0.05).

 


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of three novel hTALK-1 splice variants. TALK-1a was previously isolated from human pancreas by searching the GenBank DNA database for homologs of K2P channels (12). Here we isolated from human pancreas three splice variants of hTALK-1 denoted TALK-1b, TALK-1c, and TALK-1d. The open reading frames of TALK-1b, TALK-1c, and TALK-1d consist of 885, 969, and 789 nucleotides and encode putative proteins containing 294, 322, and 262 amino acids, respectively (Fig. 1). At the amino acid level, TALK-1b, TALK-1c, and TALK-1d are 95, 98, and 84% identical to hTALK-1a, respectively. Alignment of amino acid sequences of TALK-1a–d shows that the differences exist mainly in M4 and the COOH terminus (Fig. 1). Like TALK-1a, the three splice variants identified here each possess three N-linked glycosylation sites (residues 57, 86, and 89) located in the extracellular region between M1 and P1. TALK-1b, like TALK-1a, has a leucine zipper motif in the M4 domain. Potential phosphorylation sites for protein kinases A and C for all four variants are noted in Fig. 1. Only TALK-1b had no consensus sites for phosphorylation by either protein kinase A or C.



View larger version (61K):
[in this window]
[in a new window]
 
Fig. 1. Alignment of amino sequences of 4 splice variants of human (h) TALK-1 (GenBank accession no. AL136087 [GenBank] ). Amino acids are outlined when they are identical in at least 3 variants. Shading indicates similar amino acids. Dashes indicate gaps in alignment. Four transmembrane segments and tandem-pore regions are shown. Potential phosphorylation sites for protein kinase A and C are indicated. Three N-glycosylation sites are also present between M1 and P1 regions. A leucine zipper region in M4 segment is underlined. Splice sites are also indicated.

 

All four TALK-1 transcript variants were derived from a gene located on chromosome 6p21 (GenBank accession no. AL136087 [GenBank] ) on the basis of the comparison of cDNAs clones and genomic sequences obtained from GenBank. The TALK-1a transcript consists of six exons and five intervening regions, whereas the TALK-1b, TALK-1c, and TALK-1d transcripts consist of five exons and four intervening regions, all within the chromosomal location at 6p21.1–21.2 (Fig. 2A). As shown in Fig. 2B in more detail, TALK-1b mRNA was generated as a result of skipping of the donor splice site in the 3' end of exon 5 until a stop codon was reached. TALK-1c mRNA was formed by skipping of the entire exon 5 but including exon 6', which has an acceptor splice site located before another acceptor splice site at the beginning of exon 6. This produced a frameshift in exon 6. TALK-1d was formed by skipping of exon 5. All of the splice sites show the commonly found donor-acceptor splice consensus sequences (GT... AG) in the exonintron boundaries. Partial DNA sequences of all four TALK-1 splice variants were found in the human EST database. Thus multiple copies (>5) of TALK-1a and -1c, two copies of TALK-1b, and one copy of TALK-1d were found. Mouse TALK-1 (accession no. XM138942) was an ortholog of human TALK-1b.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2. Genomic organization and hydropathy plots of 4 splice variants of TALK-1. A: exons and intervening sequences in 4 splice variants of the hTALK-1 gene on chromosome 6. B: splice sites between exons 4 and 6; splice consensus sites are bold and overlined. C: hydropathy plots of hTALK-1 amino acid sequences using the Kyte-Doolittle algorithm show 4 putative transmembrane segments and 2 pore regions for hTALK-1a and hTALK-1b but 3 putative transmembrane segments for hTALK-1c and hTALK-1d.

 

The hydropathy plots of the four TALK-1 variants showed major differences in the COOH terminus (Fig. 2C). TALK-1b is identical to TALK-1a except for the last ~30 amino acids of the COOH terminus and has 10 fewer amino acids than TALK-1a in this region. According to the hydropathy plot, TALK-1c does not possess the fourth transmembrane segment although the COOH terminus is longer than that of TALK-1a or -1b. TALK-1d is identical in amino acid sequence to TALK-1a except that the exon 5 that encodes 53 amino acids is deleted from the M4/COOH terminus region. Therefore, only TALK-1a and TALK-1b showed complete four transmembrane segments and two pore regions.

Tissue expression of TALK-1. To confirm that mRNAs of all four TALK-1 variants are expressed in the pancreas, we carried out additional RT-PCR with primers specific to each variant. The primers were designed from DNA sequences in exons 4, 5, and 6. As shown in Fig. 3A, correct PCR products of TALK-1 variants were obtained in three separate experiments in which first-strand cDNAs were prepared with oligo(dT) as primer. Although not quantitative, these results show that all four variants are expressed in the pancreas. For TALK-1a and TALK-1d, we were able to estimate relative mRNA expressions from the density of DNA bands in the gel, as the same primers and cDNA were used under an identical condition. As shown in Fig. 3A, mRNA expression of TALK-1a was significantly (~6-fold) greater than that of TALK-1d, as judged by measurement of band intensity. However, this is a rough estimate because DNA content was not measured during the exponential phase of PCR amplification. Using the same set of primers (in exons 4 and 6) that detected TALK-1a and TALK-1d, we expected to obtain a PCR product for TALK-1c; however, a PCR product for TALK-1c was not observed. Using a different set of primers that binds to sequences within exon 6', we were able to detect a PCR product for TALK-1c (Fig. 3A).



View larger version (70K):
[in this window]
[in a new window]
 
Fig. 3. Expression of splice variants of TALK-1 in the pancreas and expression pattern of hTALK-1 mRNA in human tissues. A: RT-PCR was performed with primers specific for splice variants. Expected bands (350, 354, 381, and 207 bp for TALK-1a, -1b, -1c, and -1d, respectively) are indicated by arrowheads. DNA fragments were extracted and sequenced for confirmation. The same primers yielded TALK-1a and TALK-1d in the second lane from the left. B: Northern blot analysis was performed with the entire coding region of TALK-1b as probe and {beta}-actin as control. A major band at 2.4–3 kb is present in pancreas only.

 

The tissue expression of TALK-1a mRNA was studied previously by Northern blot analysis that showed a high level of expression in human pancreas (12). In our recent study, TASK-5 mRNA was also expressed in human pancreas and in addition it was found in other endocrine tissues such as adrenal glands (18). Therefore, we tested the possibility that TALK-1 is expressed in tissues that have not been examined previously, such as other endocrine tissues, small intestine, and stomach. Our Northern blot data showed that one or more transcripts of TALK-1 (2.4–3 kb) are present in pancreas but not in any of the other 14 tissues examined (Fig. 3). Because we used full-length TALK-1b DNA as probe and the four variants share high nucleotide identity, we expect all four variants to be detected if they are present. Our results from RT-PCR and Northern blot analyses strongly suggest that pancreas is the only major tissue that expresses TALK-1 and its variants in humans.

Functional expression of TALK-1b. The aim of this part of the study was to determine whether any splice variants of TALK-1a are capable of forming a functional channel. To determine functional expression, whole cell currents were recorded from COS-7 cells expressing a TALK-1 variant in a physiological solution containing 5 mM KCl. Cell membrane potential was held at –80 mV, and a ramp pulse was applied from –120 to +60 mV for 1 s. In cells expressing GFP alone, very small currents of <100 pA (90 ± 7 pA at +60 mV; n = 5) were recorded (Fig. 4A). In cells transfected with TALK-1a/GFP DNAs, the same voltage ramp produced a large outwardly rectifying current (723 ± 185 pA at +60 mV; n = 14), in agreement with an earlier study (12). TALK-1a current was partially inhibited by 1 mM Ba2+ (30 ± 7%; n = 7) applied to the bath solution. In cells transfected with TALK-1b/GFP DNAs, the same voltage ramp produced a similar outwardly rectifying current (721 ± 221 pA at +60 mV; n = 13) that was also partially inhibited by 1 mM Ba2+ (31 ± 8%; n = 7). In cells transfected with TALK-1c/GFP DNAs, very small currents of ~100 pA (113 ± 28 pA at +60 mV; n = 10) were recorded (Fig. 4A). Similarly, in cells transfected with TALK-1d/GFP DNAs, small currents (110 ± 16 pA at +60 mV; n = 10) not significantly different from those observed with GFP-transfected cells (control) were recorded (Fig. 4A). Figure 4B shows expressed currents of spliced variants of TALK-1 in COS-7 cells at +60 mV. These results show that TALK-1a and TALK-1b, but not TALK-1c and TALK-1d, can be functionally expressed in COS-7 cells.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4. Whole cell currents in COS-7 cells. A: whole cell currents from COS-7 cells transfected with plasmids containing DNA fragments that encode splice variants of hTALK-1. Cell membrane potential was held at –80 mV, and a ramp pulse (–120 to + 60 mV) was applied for 1 s. Ba2+ (1 mM) was applied when currents were >100 pA. GFP, green fluorescent protein. B: measured currents at +60 mV. * Significant difference from control (GFP) value (P < 0.05).

 

Figure 5A shows activation of currents after step changes in membrane potential ranging from –120 mV to +40 mV. Activation of TALK-1a and TALK-1b by step depolarization was rapid and showed no time-dependent inactivation. Ion selectivity of TALK-1b was studied by changing the concentration of K+ ([K+]) in the bath solution from 5 to 150 mM while maintaining the pipette [K+] constant at 150 mM. As shown in Fig. 5B, the reversal potential shifted to the right as [K+]in the bath solution was elevated progressively, as expected of an ion channel that is permeable to K+ but not to Cl. The plot of the reversal potentials as a function of extracellular [K+] ([K+]o) showed that the slope was 51 ± 2 mV (n = 4) per 10-fold change in [K+]o, close to the calculated Nernst value of 58 mV (Fig. 5C). These results confirm that TALK-1b is a relatively K+-selective ion channel, similar to TALK-1a (12).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5. Whole cell currents and K+ concentration ([K+]) dependence of the reversal potential. A: cell membrane potential was held at –80 mV, and currents were elicited by voltage steps from –120 to +40 mV at 20-mV intervals for 2-s duration. External [K+] was 5 mM. B: currents were elicited with ramp pulses (–120 to +60 mV, 1-s duration) applied to whole cells at different external [K+] values as indicated. C: reversal potentials (Erev) were determined at each extracellular [K+] ([K+]o) and plotted as a function of [K+]o. Experimental values were fitted by linear regression (slope 51 mV/decade).

 

The effects of various pharmacological agents were examined on the TALK-1b current with whole cell configuration from COS-7 cells. TALK-1b was insensitive to 1 mM tetraethylammonium (TEA). TALK-1b was inhibited by 1 mM Ba2+ (31 ± 8% inhibition; n = 7) and 1 mM quinidine (71 ± 8% inhibition; n = 8). Neither 4-aminopyridine (4-AP; 100 µM) nor Cs+ (1 mM) affected the TALK-1b current significantly (P > 0.05; n = 8). Arachidonic acid (20 µM), which activates K2P channels such as TREK and TRAAK (25, 30) and inhibits TASK (21, 23), failed to alter the TALK-1b current (n = 6).

Single-channel properties of TALK-1a and TALK-1b. The single-channel properties of TALK-1a have not yet been characterized in any cell system. To study the single-channel properties of TALK-1a and TALK-1b, cell-attached patches were formed on COS-7 cells transfected with plasmids containing one variant of TALK-1 and GFP. The concentration of KCl in the pipette and bath solution was 150 mM. Nearly all COS-7 cells expressing TALK-1a and TALK-1b exhibited robust channel activity that did not change significantly after 5 min (P > 0.05; n = 12). Inside-out patches showed similar channel activity, and no rundown was observed. COS-7 cells transfected with GFP alone showed no such channel activity (n > 20), consistent with the results of whole cell studies. Channel openings of TALK-1a and TALK-1b at different membrane potentials in cell-attached patches when both pipette and bath solutions contained 150 mM KCl are shown in Fig. 6, A and C. Channel openings of TALK-1a and TALK-1b were extremely brief such that the mean open time was <0.2 ms (Fig. 6, B and D). At a given membrane potential, the single-channel current amplitude of TALK-1a and TALK-1b varied markedly, producing a broad Gaussian curve in the amplitude histogram (Fig. 6, B and D). The amplitude variability of TALK-1a and TALK-1b were less at +60 mV than at –60 mV, as judged by the broadness of the histograms. The marked variability of current amplitude is likely to be due to the limitation of our recording system, because we were unable to record at a filter band pass higher than 5 kHz. Under these conditions, the estimated apparent single-channel conductances of TALK-1a and TALK-1b were 23 ± 3 and 21 ± 2 pS at –60 mV and 11 ± 2 and 10 ± 2 pS at +60 mV, respectively (n = 4). These values are underestimates of the true conductances because of the limited resolution of channel openings. The single-channel current-voltage relationships show that TALK-1a and TALK-1b are weak inwardly rectifying K+ channels in high-K+ solution (Fig. 6E). Again, our interpretation of the electrophysiological data is simply based on what we obtained under our recording conditions and may not reflect the intrinsic properties of the channel. In inside-out patches, application of 1 mM ATP or 100 µM guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) to the cytoplasmic side of the membrane produced no significant effect on TALK-1b activity (P > 0.05; n = 4 each).

Regulation of TALK-1b current by pH. TALK-1a has been shown to be activated at alkaline pH and inhibited at acidic pH (12). To study whether TALK-1b is also pH sensitive, whole cell currents of COS-7 cells expressing TALK-1b were recorded at different pHo values. TALK-1b current increased 32 ± 5% (n = 5; +60 mV) at pH 8.3 compared with that at pH 7.3 and increased further only slightly at pH 8.8 (10 ± 6%; n = 5). Similarly, TALK-1b current decreased 48 ± 6% (n = 5; +60 mV) at pH 6.3 compared with that at pH 7.3 and did not decrease further at pH 5.8 (Fig. 7A). Although not shown in Fig. 7A, TALK-1b currents at pH 6.8 and 7.8 in relation to pH 7.3 were also tested in separate cells. Averaged currents at +60 mV were determined from five experiments, normalized to the mean current determined at pH 8.8, and plotted as a function of pH (Fig. 7B). The data were fitted to a modified Boltzmann equation: y = a + {b/[1 + exp(xxo)/s]}, where a is the minimum value (0.28), b is the difference between the maximum and minimum values (0.72), x is the [H+] at which the channel activity was measured, xo is the [H+] at which half-maximal effect occurs, and s is the slope factor (0.52). At +60 mV, the half-maximal effect was observed at pH 7.2. Similar experiments were carried out on COS-7 cells expressing TALK-1a, and pH sensitivity was compared with that of TALK-1b. The results show that TALK-1a and TALK-1b possess nearly identical sensitivity to pHo near the physiological range. TALK-1c and -1d were not activated by alkaline pHo (pH 8.8), consistent with their lack of functional expression.

To study in more detail the effect of changes in pHo and pHi on TALK-1b kinetics, we recorded single-channel currents from outside-out and inside-out patches, respectively, at different pH values. Figure 7C shows channel openings from an outside-out patch kept at –60 mV in response to changes in the pHo of the bath solution from 6.3 to 8.3 at 0.5-pH unit intervals. Relative current was estimated by multiplying NPo and single-channel current amplitude at –60 mV and then plotted as a function of pHo. Single-channel current amplitudes were 1.0 ± 0.1, 1.0 ± 0.1, 1.1 ± 0.1, 1.2 ± 0.1, and 1.3 ± 0.2 pA at five pH values starting with pH 6.3. Figure 7D shows that TALK-1b is particularly sensitive to changes in pHo ranging from 6.3 to 8.3, in keeping with the whole cell current data. The single-channel amplitude was decreased only slightly (<10%) by changing the pH of the medium (Fig. 7C). These results show that the marked changes in channel activity at different pH values are largely due to apparent changes in the frequency of opening. Figure 7D also shows results obtained from inside-out patches in which the pH of the bath solution (pHi) was sequentially changed from 5.8 to 8.8. Changing pHi did not significantly affect single-channel conductance (1.0 ± 0.1 and 1.1 ± 0.1 pA at pH 5.8 and 8.8, respectively). At pH 5.8, 6.3, 8.3, and 8.8, TALK-1b currents were 75 ± 12, 84 ± 5, 120 ± 5, and 140 ± 20%, respectively, of that observed at pH 7.3. These results show that TALK-1b is much more sensitive to pHo than pHi and that the effect of pHo is not mediated via changes in pHi.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TALK-1a is a member of the K2P channel family and was first isolated from human pancreas cDNA (12). We isolated three additional splice variants of TALK-1 (TALK-1b, -1c, and -1d) from human pancreas cDNA and studied their potential functional properties. Of the three novel variants, only TALK-1b showed functional channel activity when expressed in COS-7 cells. Because of the lack of potential M4 segment in TALK-1c and -1d, we suspected that they would not form a functional channel, and indeed they showed no current in COS-7 cells. The originally cloned TALK-1a has been shown to form a functional K+ current when expressed in Xenopus oocytes and COS cells (12), and this is supported by our own data. Both whole cell and single-channel current measurements show that the two functional splice variants of TALK-1, TALK-1a and -1b, possess very similar properties with respect to their behavior as potential background K+ currents and response to various pharmacological agents.

To identify native K+ channels that are functional correlates of K2P channels, it is necessary to characterize their single-channel kinetics. We describe here the first single-channel measurement of TALK-1a and TALK-1b expressed in COS-7 cells. We (2, 15, 2022) and others (26, 29) previously described single-channel properties of functional K2P channels such as TREK, TRAAK, and TASK. The single-channel kinetics of TALK-1 is somewhat reminiscent of TRAAK kinetics as both show very short open time durations and spiky openings with variable current amplitudes. However, the two can be distinguished easily by a large difference in single-channel conductance (~22 pS for TALK-1 and 62 pS for TRAAK at –60 mV) as well as by distinctly different responses to various modulators. For example, TALK-1a and -1b are insensitive to arachidonic acid and applied pressure whereas TRAAK is activated by fatty acids and mechanosensitive (20, 26). The gating kinetics of both variants of TALK-1 is also very different from those of TREK and TASK channels. Thus the single-channel properties of TALK-1 are unique among K2P channels and perhaps among all K+ channels. This will allow us to identify a similar K+ channel in the pancreas in the future and to study its potential physiological role.

All four TALK-1 transcript variants are derived from a gene located within the human chromosome 6p21.1–21.2 region. The process of formation of four splice variants of TALK-1 is reminiscent of that of KCNK7 mRNAs that are produced by excluding exons (exon skipping) within the COOH terminus to generate several transcripts (34). In this regard, it is interesting to note that mRNA variants of several other K2P channels such as TASK-1, TREK-1, and TREK-2 are formed as a result of alternative splicing at the NH2 terminus (3, 14, 22). At present, we do not know whether TALK-1c and -1d are translated into proteins and whether they are expressed along with TALK-1a and -1b in the same pancreatic cell. If they are, the nonfunctional variants may produce a negative effect on the expression of functional TALK-1. In a preliminary test, COS-7 cells were cotransfected with plasmids containing DNAs that encode TALK-1b and TALK-1c or plasmids containing DNAs that encode TALK-1b and TALK-1d. As a control, a nonfunctional K2P channel, TASK-5 (1, 18), was coexpressed with TALK-1b. In cell-attached patches, coexpression with TALK-1c or -1d did not alter the single-channel kinetics of TALK-1b. At the whole cell level, TALK-1c or -1d also failed to alter the expression level when normalized to control. These findings suggest that TALK-1c and -1d mRNAs may not be translated into proteins or that the proteins, if synthesized, do not interact with functional forms of the variants. It is also possible that different splice variants are also expressed in distinct cell types in the pancreas. Further studies are needed to address this issue.

TALK-1a and TALK-1b share the same channel properties that define them as background K+ channels. Like other K2P channels (TASK, TREK), they both show rapid activation on voltage changes and little or no inactivation. They are open across the physiological range of membrane potentials and are generally insensitive to well-known K+ channel blockers such as TEA, 4-AP, and Cs+. However, they are blocked by high concentrations of quinidine. Although we do not yet know which types of cells in the pancreas express TALK-1a and/or -1b, these K+ channels are likely to contribute to the background K+ conductance and to regulate the resting membrane potential in those cells that express them. Islets of Langerhans in the pancreas secrete insulin, glucagon, somatostatin, and pancreatic polypeptide. The amount of hormone secretion from such cells is critically dependent on the cell membrane potential that determines Ca2+ influx and subsequently the hormone secretion. For example, insulin secretion is enhanced by increased plasma glucose concentration that elevates intracellular ATP, which in turn inhibits ATP-sensitive K+ channels. Sulfonylureas such as tolbutamide and glyburide act directly to block ATP-sensitive K+ channels. In both cases, cells depolarize because of inhibition of outward K+ flux and ultimately result in increased insulin secretion (27, 35). TALK-1 may help to regulate hormone secretion via a negative feedback mechanism, as membrane depolarization would increase TALK-1 current and drive the membrane potential toward the K+ equilibrium potential.

In addition to its putative role as a background K+ channel, TALK-1 may also serve as a signal transducer. Because TALK-1 is highly sensitive to changes in pHo, its activity would be dependent on plasma pH. Acidosis would inhibit TALK-1 current and cause depolarization, whereas alkalosis would tend to hyperpolarize the cells. Depolarization caused by acidosis would tend to enhance hormone secretion. Thus TALK-1 may have a role that is similar to those of TASK channels that are present in type I cells of carotid body and respiration-sensitive cells in the brain stem (4, 37). By sensing changes in pHo and regulating the cell membrane potential, TASK and TALK-1 channels may modulate nerve activity and hormone secretion in their respective environments. The roles of two functional splice variants of TALK-1 in hormone secretion remain to be investigated. The detailed characterization of TALK-1 variants provided here should help to identify the native K+ channel with similar properties and help to study its behavior in the native system.


    DISCLOSURES
 
This work was supported by grants from the American Heart Association and the National Institutes of Health (to D. Kim). J. Han and D. Kang are recipients of postdoctoral fellowships from the Korea Science and Engineering Foundation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. Kim, Dept. of Physiology and Biophysics, Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064 (E-mail: donghee.kim{at}finchcms.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Ashmole I, Goodwin PA, and Stanfield PR. TASK-5, a novel member of the tandem pore K+ channel family. Pflügers Arch 442: 828–833, 2001.[ISI][Medline]

2. Bang H, Kim Y, and Kim D. TREK-2, a new member of the mechanosensitive tandem-pore K+ channel family. J Biol Chem 275: 17412–17419, 2000.[Abstract/Free Full Text]

3. Bockenhauer D, Zilberberg N, and Goldstein SA. KCNK2: reversible conversion of a hippocampal potassium leak into a voltage-dependent channel. Nat Neurosci 4: 486–491, 2001.[ISI][Medline]

4. Buckler KJ, Williams BA, and Honore E. An oxygen-, acid- and anaesthetic-sensitive TASK-like background potassium channel in rat arterial chemoreceptor cells. J Physiol 525: 135–142, 2000.[Abstract/Free Full Text]

5. Chavez RA, Gray AT, Zhao BB, Kindler CH, Mazurek MJ, Mehta Y, Forsayeth JR, and Yost CS. TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family. J Biol Chem 274: 7887–7892, 1999.[Abstract/Free Full Text]

6. Czirjak G and Enyedi P. Formation of functional heterodimers between the TASK-1 and TASK-3 two-pore domain potassium channel subunits. J Biol Chem 277: 5426–5432, 2002.[Abstract/Free Full Text]

7. Czirjak G and Enyedi P. TASK-3 dominates the background potassium conductance in rat adrenal glomerulosa cells. Mol Endocrinol 16: 621–629, 2002.[Abstract/Free Full Text]

8. Decher N, Maier M, Dittrich W, Gassenhuber J, Bruggemann A, Busch AE, and Steinmeyer K. Characterization of TASK-4, a novel member of the pH-sensitive, two-pore domain potassium channel family. FEBS Lett 492: 84–89, 2001.[ISI][Medline]

9. Duprat M, Lesage F, Fink M, Reyes R, Heurteaux C, and Lazdunski M. TASK, a human background K+ channel to sense external pH variations near physiological pH. EMBO J 16: 5464–5471, 1997.[Abstract/Free Full Text]

10. Fink M, Duprat F, Lesage F, Reyes R, Romey G, Heurteaux C, and Lazdunski M. Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel. EMBO J 15: 6854–6862, 1996.[Abstract]

11. Fink M, Lesage F, Duprat F, Heurteaux C, Reyes R, Fosset M, and Lazdunski M. A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids. EMBO J 17: 3297–3308, 1998.[Abstract/Free Full Text]

12. Girard C, Duprat F, Terrenoire C, Tinel N, Fosset M, Romey G, Lazdunski M, and Lesage F. Genomic and functional characteristics of novel human pancreatic 2P domain K+ channels. Biochem Biophys Res Commun 282: 249–256, 2001.[ISI][Medline]

13. Goldstein SAN, Bockenhauer D, and Zilberberg N. Potassium leak channels and the KCNK family of two-P-domain subunits. Nat Neurosci 2: 175–184, 2001.

14. Gu W, Schlichthörl G, Hirsch JR, Engels H, Karschin C, Karschin A, Derst C, Steinlein OK, and Daut J. Expression pattern and functional characteristics of two novel splice variants of the two-pore-domain potassium channel TREK-2. J Physiol 539: 657–668, 2002.[Abstract/Free Full Text]

15. Han J, Truell J, Gnatenco C, and Kim D. Characterization of four types of background potassium channels in rat cerebellar granule neurons. J Physiol 542: 431–444, 2002.[Abstract/Free Full Text]

16. Honore E, Maingret F, Lazdunski M, and Patel AJ. An intracellular proton sensor commands lipid- and mechano-gating of the K+ channel TREK-1. EMBO J 21: 2968–2976, 2002.[Abstract/Free Full Text]

17. Kim D and Clapham DE. Potassium channels in cardiac cells activated by arachidonic acid and phospholipids. Science 244: 1174–1176, 1989.[ISI][Medline]

18. Kim D and Gnatenco C. TASK-5, a new member of the tandem-pore K+ channel family. Biochem Biophys Res Commun 284: 923–930, 2001.[ISI][Medline]

19. Kim D, Sladek CD, Aquado-Velasco C, and Mathiasen JR. Arachidonic acid activation of a new family of K+ channels in cultured rat neuronal cells. J Physiol 484.3: 643–660, 1995.[Abstract]

20. Kim Y, Bang H, Gnatenco C, and Kim D. Synergistic interaction and the role of C-terminus in the activation of TRAAK K+ channels by pressure, free fatty acids and alkali. Pflügers Arch 442: 64–72, 2001.[ISI][Medline]

21. Kim Y, Bang H, and Kim D. Task-3, a new member of the tandem pore K+ channel family. J Biol Chem 275: 9340–9347, 2000.[Abstract/Free Full Text]

22. Kim Y, Bang HW, and Kim D. TBAK-1 and TASK-1, two-pore K+ channel subunits: kinetic properties and expression in rat heart. Am J Physiol Heart Circ Physiol 277: H1669–H1678, 1999.[Abstract/Free Full Text]

23. Kim Y, Gnatenco C, Bang H, and Kim D. Localization of TREK-2 K+ channel domains that regulate channel kinetics and sensitivity to pressure, fatty acids and pHi. Pflügers Arch 442: 952–960, 2001.[ISI][Medline]

24. Lesage F, Guillemare E, Fink M, Duprat F, Lazdunski M, Romey G, and Barhanin J. TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure. EMBO J 15: 1004–1011, 1996.[Abstract]

25. Lesage F and Lazdunski M. Molecular and functional properties of two-pore-domain potassium channels. Am J Physiol Renal Physiol 279: F793–F801, 2000.[Abstract/Free Full Text]

26. Maingret F, Fosset M, Lesage F, Lazdunski M, and Honore E. TRAAK is a mammalian neuronal mechano-gated K+ channel. J Biol Chem 274: 1381–1387, 1999.[Abstract/Free Full Text]

27. Nichols CG and Koster JC. Diabetes and insulin secretion: whither KATP? Am J Physiol Endocrinol Metab 283: E403–E412, 2002.[Abstract/Free Full Text]

28. Patel AJ and Honore E. Properties and modulation of mammalian 2P domain K+ channels. Trends Neurosci 24: 339–346, 2001.[ISI][Medline]

29. Patel AJ, Honore E, Maingret F, Lesage F, Fink M, Duprat F, and Lazdunski M. A mammalian two pore domain mechano-gated S-like K+ channel. EMBO J 17: 4283–4290, 1998.[Abstract/Free Full Text]

30. Patel AJ, Lazdunski M, and Honore E. Lipid and mechano-gated 2P domain K+ channels. Curr Opin Cell Biol 13: 422–428, 2001.[ISI][Medline]

31. Patel AJ, Maingret F, Magnone V, Fosset M, Lazdunski M, and Honore E. TWIK-2, an inactivating 2P domain K+ channel. J Biol Chem 275: 28722–28730, 2000.[Abstract/Free Full Text]

32. Pountney DJ, Gulkarov I, Vega-Saenz de Miera E, Holmes D, Saganich M, Rudy B, Artman M, and Coetzee WA. Identification and cloning of TWIK-originated similarity sequence (TOSS): a novel human 2-pore K+ channel principal subunit. FEBS Lett 450: 191–196, 1999.[ISI][Medline]

33. Rajan S, Wischmeyer E, Karschin C, Preisig-Muller R, Grzeschik KH, Daut J, Karschin A, and Derst C. THIK-1 and THIK-2, a novel subfamily of tandem pore domain K+ channels. J Biol Chem 276: 7302–7311, 2001.[Abstract/Free Full Text]

34. Salinas M, Reyes R, Lesage F, Fosset M, Heurteaux C, Romey G, and Lazdunski M. Cloning of a new mouse two-P domain channel subunit and a human homologue with a unique pore structure. J Biol Chem 274: 11751–11760, 1999.[Abstract/Free Full Text]

35. Sharma N, Crane A, Gonzalez G, Bryan J, and Aguilar-Bryan L. Familial hyperinsulinism and pancreatic beta-cell ATP-sensitive potassium channels. Kidney Int 57: 803–808, 2000.[ISI][Medline]

36. Talley EM and Bayliss DA. Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9) potassium channels: volatile anesthetics and neurotransmitters share a molecular site of action. J Biol Chem 277: 17733–17742, 2002.[Abstract/Free Full Text]

37. Washburn CP, Sirois JE, Talley EM, Guyenet PG, and Bayliss DA. Serotonergic raphe neurons express TASK channel transcripts and a TASK-like pH- and halothane-sensitive K+ conductance. J Neurosci 22: 1256–1265, 2002.[Abstract/Free Full Text]