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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Physiology and Biophysics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois; and 2Department of Physiology, College of Medicine and Institute of Health Science, Gyeongsang National University, Jinju, Korea
Submitted 15 December 2005 ; accepted in final form 15 February 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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two-pore domain K+ channel; conductance; excitability
In addition to their role as background K+ channels, K2P channels are likely to contribute to other important biological processes, as they are sensitive to various biologically important stimuli. For example, members of the TREK/TRAAK subfamily are sensitive to temperature, mechanical force, lipids, and acid/alkali (14, 17, 26). Some K2P channels such as TREKs and TASKs are activated by volatile anesthetics and may contribute to anesthetic-induced hyperpolarization (25). TREKs and TASKs are also strongly inhibited by various neurotransmitters via G protein-coupled receptors, providing an important signaling pathway for modulating cellular excitability and synaptic transmission (3, 7, 32). Members of the TALK family are particularly sensitive to changes in pH in the alkaline range (10). Thus modulation of these background K+ channels by diverse physiological factors will lead to alterations in cell excitability.
The goal of this study was to identify and characterize background K+ channels in DRG neurons, determine their molecular correlate, and find out which K+ channels provide the major background K+ conductance in these neurons. Therefore, we recorded single-channel openings from cell-attached and inside-out patches from cultured DRG neurons with small- and medium-sized cell bodies and compared the contribution of each K+ channel to the resting K+ current. Because TREK channels are thermosensitive and are activated by heat (9, 14), experiments were done at both 24°C and 37°C. Our study shows that although many K+ channels including K2P channels are expressed in the cell body of DRG neurons, TRESK and TREKs serve as the major background K+ channels. As these K2P channels are highly modulated by various factors including G proteins, they are likely to be important regulators of cell excitability in DRG neurons under various physiological and pathophysiological conditions.
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METHODS |
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DRG neuron culture. Cultured DRG neurons were prepared as described previously (24). All animals were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23). The animal protocol was approved by Institutional Animal Care and Use Committee at Rosalind Franklin University. Briefly, DRGs were dissected from thoracic and lumbar levels of the spinal cord of 1- or 2-day-old neonatal rats (total number of rats used was 22). DRGs were collected in cold (4°C) DMEM-F-12 medium (50:50 vol/vol) containing fetal bovine serum (10%; Life Technologies), 1 mM sodium pyruvate, 25 ng/ml nerve growth factor (Sigma, St. Louis, MO), and 100 U/ml of penicillin-streptomycin (Sigma). Ganglia were washed three times with DMEM-F-12 medium and incubated for 30 min in DMEM-F-12 medium containing 1 mg/ml collagenase (type II; Worthington, Freehold, NJ). The ganglia were then washed three times with Mg2+- and Ca2+-free HBSS and incubated with gentle shaking in warm (37°C) HBSS containing 2.5 mg/ml trypsin (Life Technologies). The solution was centrifuged at 1,000 rpm for 10 min, and the pellet was washed three times with the culture medium to inhibit the enzyme. The pellet was suspended in the culture medium and gently triturated with a heat-polished Pasteur pipette. The suspension was plated on glass coverslips treated with poly-L-lysine and placed in a 24-well culture dish. Cells were incubated at 37°C in a 95% air-5% CO2 gas mixture. Cells were used 1–5 days after being plated.
RT-PCR analysis. First-strand cDNAs were synthesized from total RNA isolated from DRG neurons individually picked from DRG neuronal culture and spinal cord with oligo(dT) (Superscript preamplification system, Invitrogen, Rockville, MD). Mouse testis total RNA was purchased from Stratagene, and cDNA was prepared similarly. First-strand cDNA was used as a template for PCR amplification. Specific primers for each K2P channel were used for PCR with Taq polymerase (Takara). Table 1 lists the DNA sequences of primers used to detect expression of each K2P channel. PCR conditions were initial denaturation at 94°C for 4 min, then 30–35 cycles at 94°C for 45 s, 55–63°C for 1 min, 72°C for 2 min, and a final extension step at 72°C for 10 min. For TRESK, the annealing temperature was 63°C (35 cycles). For TREK-2 and TASKs, the annealing temperature was 55°C (30 cycles). For TREK-1 and TRAAK, the annealing temperature was 55°C (20 s, 30 cycles). The DNA fragments obtained by RT-PCR with DRG neurons, spinal cord, and mouse testis were subcloned into pcDNA3.1 TOPO vector by TA cloning. Subcloned DNA fragments were sequenced on both strands with the dideoxynucleotide chain termination method.
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RESULTS |
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The number of patches showing one type of K+ channel was determined and plotted in Fig. 1B as a percentage value that should indicate the relative abundance of the K+ channel, the tracing of which is shown in Fig. 1A. The relative current was also calculated from the equation IREL = (NPoi)(% of patches observed), where i is the single-channel amplitude at +60 or –60 mV. NPo was determined from a large number of recordings (>24 for each channel type) and averaged to obtain NPo per patch. The results shown in Fig. 1C clearly identify the 50-pS channel as the major background K+ channel at 37°C, with the 14- and 112-pS channels contributing to a smaller extent. However, at 24°C (room temperature), the relative currents provided by the four K+ channels shifted such that the 14-pS channel became the major active channel. The total number of patches used was 72 at 24°C. The other three K+ channels (50, 112, and 73 pS) showed low activity at 24°C, due to the temperature dependence of these channels (see below). These findings point to the importance of studying such channels at a physiological temperature. When the membrane potential was held at –60 mV to record inward current, the relative currents contributed by each of the four K+ channels were similar to those observed at +60 mV (Fig. 1C). Together, the 14-pS, 50-pS, and 112-pS channels contributed 
97% of the current provided by all four channels at 37°C. Among the four K+ channels, the 50-pS channel was the most active at 37°C.
Molecular identification of 14-pS channel as TRESK.
Among the K+ channels shown in Fig. 1A, the most frequently observed and thus most abundantly expressed channel was the 14-pS channel. The percentage of the 14-pS channel expressed in small- and medium-diameter neurons was 40% and 42%, respectively, indicating that the 14-pS channel is evenly distributed in these neurons. In 11 neurons with cell body diameter larger than 25 µm (26–38 µm), the 14-pS channel was also observed in 5 cells, indicating that the 14-pS channel is present in DRG neurons with large-diameter cell bodies. To test for ion selectivity, K+ concentration in the bath solution was lowered from 150 to 30 mM KCl. This caused a shift in the reversal potential from zero to +35 ± 3 mV, as expected of a K+-selective, but not a Cl–-selective, ion channel. No inward current was present when the pipette K+ was replaced with choline or Na+, indicating that the ion channel was not a nonselective cation channel. The single-channel conductance was 16 ± 2 pS at –60 mV and 14 ± 1 pS at +60 mV (n = 7), producing a nearly linear current-voltage relationship. Under our recording conditions using pipettes with 4-M
tip resistance, one to three channels were usually present in the patches, as judged by the number of open levels. The 14-pS channel remained active after formation of inside-out patches and showed no sign of desensitization or rundown at either +60 or –60 mV. Thus the 14-pS channel is clearly a background K+ channel in DRG neurons.
To test whether the 14-pS DRG K+ channel is a member of the K2P channel family, we compared the single-channel kinetics of this native K+ channel with those of cloned K2P channels expressed in COS-7 cells. Of the 10 K2P channels examined, only TRESK and TASK-1 showed conductance of 14 pS in 150 mM KCl (–60 mV). TASK-1, however, exhibited rapid kinetics, with a mean open time of 1 ms and an inwardly rectifying current-voltage relationship, characteristics that are clearly distinct from those of the 14-pS channel, which shows a long open state at depolarized potentials and a linear current-voltage relationship. Therefore, we tested the possibility that the 14-pS channel is encoded by TRESK by comparing the biophysical and pharmacological properties of the two K+ channels in more detail. Single-channel openings of the DRG 14-pS K+ channel and mouse TRESK expressed in COS-7 cells recorded at various membrane potentials in cell-attached patches are shown in Fig. 2A. The single-channel conductance of TRESK expressed in COS-7 cells was 16 ± 2 pS at –60 mV and 14 ± 1 pS at +60 mV (n = 5), similar to that of the 14-pS channel in DRG neurons. Both TRESK and the 14-pS DRG neuron K+ channel showed nearly linear current-voltage relationships (Fig. 2B). The 14-pS DRG channel and TRESK showed similar low sensitivity to changes in voltage between –60 and +60 mV. At more depolarized potentials (+80 mV), channel activity was slightly higher (Fig. 2C).
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Molecular identification of 112-pS and 50-pS channels as TREK-1 and TREK-2. The open probability of the 112-pS and 50-pS channels was generally low at room temperature (Po < 0.005), as described in Fig. 1. However, these channels became active at 37°C, allowing clear characterization of their single-channel kinetics. The percentages of patches in which these channels were recorded from a total of 276 patches were 11% and 25% for 112-pS and 50-pS channels, respectively (Fig. 1B). The 112-pS and 50-pS DRG channels were K+ selective, as the reversal potential shifted to the right by 36 ± 3 and 35 ± 4 mV, respectively, when bath KCl concentration was lowered from 150 to 30 mM in inside-out patches. Figure 4, A and D, show side-by-side comparisons of single-channel openings of the two DRG K+ channels with TREK-1 and TREK-2 expressed in COS-7 cells. The single-channel conductance and current-voltage relationships of two native K+ channels (112 ± 7 and 50 ± 7 pS at +60 mV) were similar to those of cloned TREK-1 (115 ± 6 pS) and TREK-2 (49 ± 8 pS), respectively, expressed in COS-7 cells (Fig. 4, B and E). DRG K+ channels and TREKs also share an unusually high open channel noise observed at both positive and negative membrane potentials.
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Molecular identification of 73-pS channel as TRAAK. The 73-pS channel in cell-attached patches of DRG neurons with 150 mM KCl in the pipette and bath solutions at 37°C is shown in Fig. 5A. The 73-pS channel showed spikelike openings to various levels, due to limited resolution of the recording system. This is similar to those observed with TRAAK, as shown by the comparison of single-channel openings at positive and negative potentials (Fig. 5A). The mean open times were too short (<0.3 ms) to be accurately measured for both the 73-pS channel and TRAAK expressed in COS-7 cells. To determine the single-channel amplitude, only the maximally open channels were used, as illustrated by the dotted lines in Fig. 5A. Under this condition, the current-voltage relationships of the 73-pS channel and TRAAK expressed in COS-7 cells could be superimposed (Fig. 5B). Thus the kinetic properties of the 73-pS channel are similar to those of TRAAK. No other K2P channels showed such channel kinetics and conductance.
More helpful evidence for identifying the molecular correlate of the 73-pS channel came from studies that tested its modulation by various stimuli. Like TREKs, TRAAK is strongly activated by negative pressure and arachidonic acid (18). However, TRAAK is activated by alkaline conditions and is insensitive to intracellular acid. To further test whether the 73-pS channel is similar to TRAAK, we studied the effect of applying negative pressure, arachidonic acid, and acid/alkali solution on the 73-pS channel. In inside-out patches from DRG neurons containing the 73-pS channel, application of –20 mmHg pressure or arachidonic acid to the bath solution elicited large increases in channel activity (Fig. 5C). When acid solution (pH 6.0) was applied to the bath solution, no significant effect was observed in channel activity. However, application of alkali solution (pH 8.3) caused a marked increase in channel activity, similar to that observed with TRAAK expressed in COS-7 cells (Fig. 5C). Heating the bath solution to 37°C from 24°C produced an increase in channel activity of both the 73-pS channel and TRAAK. Thus the biophysical and pharmacological properties of the 73-pS channel indicate that it is most likely encoded by TRAAK.
Other backgroundlike K+ channels with low open probability in DRG neurons. In addition to the four DRG K+ channels described above, three other K+ channels with single-channel conductance of 16–36 pS (9 patches), 33–34 pS (8 patches), and 34–69 pS (2 patches) were also recorded from 276 cell-attached patches at +60 and –60 mV with pipette solution containing 150 mM KCl. These channels were constitutively open at holding potentials of +60 and –60 mV and did not desensitize in cell-attached patches. The frequency of observing these channels was too low to clearly characterize and identify their molecular counterpart, particularly when other large-conductance channels like TREKs were also present in the same patch. All three channels were determined to be K+ selective, as judged by a +30- to +36-mV shift in the reversal potential from zero when intracellular KCl was changed from 150 mM to 30 mM and by the lack of outward channel activity when K+ in the bath was replaced with Na+ (n = 3–5). The relative current contributed by the three K+ channels together at +60 mV at 37°C, determined by the same method as above (Fig. 1), was estimated to be 0.01 (1.3%), compared with 0.52 (69%) by TREK-2 and 0.12 (16%) by TRESK. Therefore, the three backgroundlike K+ channels with low open probability and low expression levels are expected to play a very minor role in cell excitability under normal resting conditions.
DRG neurons in culture express TRESK and TREK/TRAAK mRNAs.
The functional studies described above suggest that DRG neurons should express mRNAs for TRESK and TREK/TRAAK. In our recent study (15), TRESK cDNA was cloned from mouse testis and Northern blot analysis showed that the expression of TRESK mRNA was positive in many tissues but generally low, as judged by weak signals. To determine whether TRESK mRNA is expressed in DRG neurons, we performed RT-PCR analysis using the first-strand cDNA synthesized from 800 cell bodies of DRG neurons picked from cultured cells. PCR fragment of TRESK was clearly detected in the testis as reported earlier (15) (Fig. 6). A relatively strong signal was also detected for DRG neurons (Fig. 6). Positive signal was also detected in the rat spinal cord. In addition to TRESK mRNA, DRG neurons expressed mRNAs for TREK-1, TREK-2, and TRAAK, as expected from our functional studies. TASK-1 and TASK-3 mRNAs were also detected in DRG neurons.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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–60 mV (27, 34). This negative resting membrane potential is likely to be the net effect of both the inward current via the nonselective cation channels and the larger outward current via K+ channels. The nature of the K+ channels that give rise to the resting K+ conductance in DRG neurons is not well known. Because mRNA species of many K2P channels are expressed in DRG neurons (21) and these K2P channels have properties of background K+ channels, it seems reasonable to speculate that some of these K+ channels would contribute to the background K+ conductance in DRG neurons. However, the measured levels of mRNAs do not always correlate with the levels of surface functional protein products. Therefore, the goal of this study was to define the properties of background (leak, resting) K+ channels expressed in DRG neurons, determine the molecular identity of each significant K+ channel, and show which K2P channels provide the major background K+ conductance. By recording single channels in cell-attached patches and characterizing the biophysical and pharmacological properties, we show that cell bodies of small (diameter 10–16 µm)- to medium (diameter 17–25 µm)-sized DRG neurons express four major types of K+ channels, all of which belong to the K2P channel family. These four K+ channels are TRESK (K2P18.1), TREK-1 (K2P2.1), TREK-2 (K2P10.1), and TRAAK (K2P4.1). The relative functional expression level of each channel type, as judged by the number of channels recorded from many patches, and measurement of their averaged channel activities show that TREK-2 would provide the major background K+ conductance in cell body of small- to medium-sized DRG neurons at 37°C. However, at lower temperatures, the relative contribution by TRESK increases, and it is the major background K+ channel at 24°C. Because these K2P channels are modulated by various biological stimuli including agonists that act on G protein-coupled receptors (3, 8), they are expected to play an active role in the regulation of excitability of DRG neurons.
Functional expression of TRESK in DRG neurons.
The most effective way to identify and characterize individual background K+ channels is by recording single-channel openings from cell-attached patches with high KCl in the pipette solution. Using this method, we were able to identify a small-conductance channel (14 pS) in 41% of the patches formed on DRG neurons. The 14-pS channel was active at rest and showed no time-dependent inactivation or desensitization, and the amount of current was a function of how far the membrane potential was held from the reversal potential. These properties are consistent with it being a background K+ channel. A K2P channel with properties similar to those of the 14-pS channel was previously described as TRESK (15, 31). Detailed comparisons of the biophysical and pharmacological properties of this native 14-pS K+ channel with those of TRESK expressed in COS-7 cells suggest strongly that they are the same channel protein. Furthermore, single-channel analysis of all functional K2P channels showed that TRESK is the only K channel that has single-channel kinetics and conductance similar to those of the native 14-pS channel. TRESK was reported to be highly sensitive to Zn2+ and Hg2+ (6), and this should further help to identify native TRESK in the future. However, differences in the sensitivity of human and rodent TRESK to various pharmacological modulators (Zn2+, anesthetic agents) must be carefully considered before identifying the native TRESK (16). Our present study is the first to show that TRESK is functionally expressed and acts as a background K+ channel in the native system.
The original TRESK was first cloned from human spinal cord (31). A mouse TRESK with 63% amino acid identity with human TRESK was then cloned from mouse cerebellar RNA by RT-PCR (8). In our recent study (15), mouse TRESK was also cloned from mouse testis. We named it TRESK-2 because the first 544 bp of this mouse clone could be isolated from human spinal cord and testis cDNA (purchased from Stratagene and OriGene) with RT-PCR. Therefore, we presumed that human tissue expresses two TRESK isoforms (15). However, search of the human genome and expressed sequence tag (EST) database for the mouse TRESK sequence did not yield any positive results. Furthermore, the human TRESK DNA sequence could not be found in the mouse/rat EST library. Our RT-PCR studies show that only the mouse TRESK (which we previously named TRESK-2) is present in rat DRG neurons. Therefore, there is yet no clear indication for the existence of two TRESK isoforms, and human and mouse TRESK will be assumed to be orthologs. Human TRESK and mouse (and rat) TRESK exhibit identical single-channel behavior and sensitivity to pharmacological agents tested so far.
Functional expression of TREK/TRAAK in DRG neurons. By recording single-channel openings in cell-attached patches from DRG neurons, we were also able to identify three other K+ channels (112, 50, and 73 pS) with properties similar to those of TREK-1, TREK-2, and TRAAK, which are three closely related members of the TREK subfamily. This identification is based on their single-channel kinetics and high sensitivity to arachidonic acid, membrane stretch, and acid/alkali, as we have done recently in other cell types (12, 13). Because well-known K+ channel blockers, such as tetraethylammonium, apamin, aminopyridine, and Cs+, at concentrations that normally block other voltage-gated K+ channels, have little effects on these DRG K+ channels, the lack of action of these agents on the 112-pS, 50-pS, and 73-pS channels was also helpful in their molecular identification.
Our results show that TRESK is the most active K+ channel at rest at room temperature, contributing 92% of the resting K+ current. This is because TREK/TRAAK are relatively inactive at room temperature, although they can be strongly activated by free fatty acids, negative pressure, and acid/alkali. Recent studies show that all three K+ channels are thermosensitive (14). Thus, when experiments were repeated at 37°C, TREK-2 was found to be the most active background K+ channel, followed by TRESK, TREK-1, and TRAAK. Our results also show that although mRNA transcripts of many K2P channels are expressed in DRG neurons (21), only a few provide a significant contribution to the overall background K+ conductance. Therefore, measurement of channel mRNA expression cannot always be used to estimate functional expression levels. In keeping with this view, TASK-1 current in rat ventricular myocytes was found to be very low, despite a strong mRNA expression as measured by Northern blot and RT-PCR analyses in these cells (19).
DRG neurons express mRNA transcripts for TASK-1, TASK-2, and TASK-3 (21), suggesting that they could be the molecular correlates of the three low-activity K+ channels that we observe in cell-attached patches. However, we have been unable to confirm this, because of the low expression and low channel activity of the native K+ channels. In any case, TASK-like channels do not appear to provide significant background K+ current, at least in the cultured DRG neurons used in this study. In DRG neurons from adult rat, small TASK-like K+ currents were recorded at the whole cell level (5), suggesting that TASK expression may be higher in adult than in neonatal rat DRG neurons used in this study.
Another ion channel that was observed in cell-attached patches of DRG neurons was a 33-pS-conductance channel with a linear current-voltage relationship. Because of the relatively low level of expression, this 33-pS K+ channel is also unlikely to contribute much to the background K+ conductance in DRG neurons. On the basis of averaged channel activities and single-channel conductance, the 33-pS channel would contribute <1% of that provided by TRESK-2/TREKs. Other K2P channels such as TWIK-1 are also expressed in DRG neurons at the mRNA level (21). However, we did not detect any TWIK-1-like channels described recently, possibly because of its sumoylated state that silences this channel (28).
Physiological implications of TRESK and TREKs as background K+ channels. Nerve terminals of DRG neurons are located within the dermal and epidermal layers of the skin; sense various physical and chemical stimuli such as heat, cold, and touch; and transmit these signals to the brain via the spinal tracts. Whether TRESK and TREK/TRAAK are expressed in these sensory nerve terminals remains to be determined. If they were present, the relative contribution of TRESK and TREKs would vary depending on the temperature near the skin, because of the temperature sensitivity of TREKs. Transient receptor potential (TRP) ion channels are considered to be the major cellular sensors of such stimuli, as these nonselective cation channels are sensitive to temperature, touch, cell volume, and taste (2, 4). Activation of specific TRP ion channels by a stimulus increases influx of cations, causing depolarization and increased excitability of the neurons involved. In our previous study (24), nearly all small-sized DRG neurons expressed the capsaicin-sensitive nonselective cation channel TRPV1. The functional presence of TRESK/TREKs and TRP ion channels in the same neurons suggests that both types of ion channels would be involved in the regulation of cell excitability in response to various stimuli. Cold temperature has been shown to not only activate a TRP ion channel (TRPM8) but also inhibit the background K+ current, both of which would contribute to depolarization and increased excitability (1, 30, 33).
Agonists that act on Gq/11-coupled receptors are well known to inhibit TREKs (3, 22). This suggests that the DRG background K+ current that is significantly contributed by TREKs is an important regulator of cell excitability. Transmission of sensory information by DRG neurons may therefore involve inhibition of TREKs by local neurotransmitters and subsequently increased excitability. Interestingly, a recent study suggests that receptor agonists that elevate intracellular Ca2+ concentration activate TRESK via dephosphorylation by calcineurin (8). Although we were unable to directly test the effect of agonists on the 14-pS channel in DRG neurons because of the presence of other ion channels, we found that TRESK expressed in COS-7 cells were activated (42% increase) by acetylcholine via coexpressed M3 receptors. Because stimulation of M3 receptors coupled to Gq/11 leads to elevated cytoplasmic Ca2+ levels, an agonist that stimulates Gq/11 would be expected to inhibit TREKs and activate TRESK in the same neuron. Perhaps TRESK and TREKs and the associated receptors are expressed in different regions of the DRG neurons and mediate signaling by different agonists to finely modulate cell excitability. It would be important to test such possibilities in the future.
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FOOTNOTES |
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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.
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