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Single-channel Recordings of TREK-1 K⁺ Channels in Periodontal Ligament Fibroblasts

¹ Department of Bioscience,
² Department of Basic Science for Health and Nursing,
³ Department of Oral and Maxillofacial Surgery, and
⁴ Department of Legal Medicine, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan

ohara{at}belle.shiga-med.ac.jp

	ABSTRACT

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The periodontal ligament (PDL) works as a suspensory ligament when external mechanical stress is placed on the teeth. PDL fibroblasts, the principal cells in the PDL, are responsible for many PDL functions. We hypothesized that mechanosensitive ion channels are present in human PDL fibroblasts, which are capable of responding to mechanical stress during normal function of the tissue. Using patch-clamp techniques, we detected mechanosensitive TREK-1 K⁺ channels (a member of the two-pore-domain K⁺ channel family), whose single-channel conductance was 104 pS in symmetrical K⁺-rich solutions. The open probability of the channel was low in the quiescent state, but it was strongly increased by the induction of membrane stretch. Arachidonic acid also enhanced the channel activity. RT-PCR and immunocytochemical observations showed the expression of TREK-1 K⁺ channels in PDL fibroblasts. The results suggest that the activation of TREK-1 K⁺ channels by masticatory stress contributes to the hyperpolarization of PDL fibroblasts.

KEY WORDS: TREK-1 • K⁺ channel • periodontal ligament • teeth • single-channel recording

	INTRODUCTION

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The periodontal ligament (PDL) is a highly specialized tissue that serves to attach the teeth to the supporting bone, and functions as a cushion that relieves masticatory forces. The principal cell in PDL is the periodontal ligament fibroblast (PDL fibroblast), which is responsible for many PDL functions, such as support of teeth, formation and maintenance of connective fibers, and regeneration of the PDL itself. There have been many cytological, biochemical, and physiological studies on PDL fibroblasts, but few studies have investigated ion channels present in PDL fibroblasts. To our knowledge, there has been no study to clarify single-channel properties of ion channels in PDL fibroblasts by patch-clamp techniques.

The TREK-1 K⁺ channel (TWIK-related K⁺ channel), which is a member of the two-pore-domain K⁺ channel family, is widely expressed in many kinds of tissues, including brain, spinal cord, lung, kidney, small intestine, heart, and skeletal muscle (Lesage and Lazdunski, 2000a; Medhurst et al., 2001; O’Connell et al., 2002). Since the TREK-1 K⁺ channel is opened by various physical and chemical stimuli—including mechanical stress (Patel et al., 1998), temperature (Maingret et al., 2000a), intracellular acidosis (Maingret et al., 1999), and polyunsaturated fatty acids (Maingret et al., 2000b)—the channel is involved in many cellular functions, including regulation of nerve excitability (Bockenhauer et al., 2001), ischemic neuroprotection (Lauritzen et al., 2000; Buckler and Honoré, 2005; Caley et al., 2005), and heterogeneous repolarization of cardiomyocytes (Tan et al., 2004). In dental tissues, it has been recently reported that TREK-1 K⁺ channels are expressed in human odontoblasts, and that odontoblasts transmit sensory signals evoked by mechanical stress to the nerve terminus via the activation of TREK-1 K⁺ channels (Magloire et al., 2003).

Since PDF fibroblasts, as well as odontoblasts, are differentiated from neural crest cells, PDL fibroblasts may have the same cellular mechanisms for response to mechanical stress. In this study, we hypothesized that mechanosensitive ion channels are present in human PDL fibroblasts, which are capable of responding to mechanical stress during normal function of the tissue.

	MATERIALS & METHODS

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Cell Culture
Human PDL fibroblasts were obtained from healthy erupted third molars, which were extracted for orthodontic reasons, with the informed consent of the patients. The study protocol was approved by the Ethical Committee of the Shiga University of Medical Science. The extracted teeth were rinsed several times with the culture medium, Dulbecco’s Modified Eagle’s Medium (DMEM), containing 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin. Periodontal ligament (PDL) was dissected from the middle third of the tooth root, according to the primary culture method of PDL fibroblasts (Somerman et al., 1988). The dissected pieces were placed in 3.5-cm culture dishes, and were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂. When the cells, which had migrated from the explants, became confluent, the cultures were trypsinized with phosphate-buffered saline containing 0.25% trypsin and 1 mM EDTA, and the cells were transferred to 10-cm dishes. The cells that reached confluence were defined as the first passage, and the cells at the second passage were stored in liquid nitrogen for use in subsequent experiments. It has been shown that the cells obtained by the above procedure have properties of PDL fibroblasts (Nishikawa et al., 2002). The cells between the third and the fifth passages, after 10- to 14-day culture, were used for the experiments.

Patch-clamp Measurements
PDL fibroblasts were seeded on 3.5-cm plastic dishes for patch-clamp experiments. Patch pipettes were made of WPI TW150 glass (New Haven, CT, USA), pulled with a PP-830 patch-pipette puller (Narishige, Tokyo, Japan), and heat-polished to produce tip diameters of about 1 µm with about 10 M resistance when filled with pipette solutions. The pipette solution contained 100 nM iberiotoxin (Peptide Institute Inc., Minoh, Japan) to avoid the contamination of the current records with BK potassium channel currents. The extracellular solution (high-Na⁺ solution) for patch-clamp experiments contained (in mM) 136 NaCl, 5 KCl, 2 CaCl₂, 1.5 MgCl₂, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at pH 7.4, adjusted with about 4 NaOH. The intracellular solution (high-K⁺ solution) contained (in mM) 10 NaCl, 110 KCl, 2.69 ,N'-CaCl₂, 5 ethylene glycol-bis(b-aminoethyl ether)-N,N,N' tetraacetic acid (EGTA) (10^–7 M free Ca²⁺), 2 MgCl₂, and 10 HEPES at pH 7.4, adjusted with about 24 KOH. Single-channel currents were recorded in cell-attached and inside-out patches with a CEZ-2300 amplifier (Nihon Kohden, Tokyo, Japan). To examine the effects of mechanical stress on the activity of TREK-1 channels, we elicited membrane stretch by applying suction (negative pressure) through a side port of the pipette holder to the patch membrane. The pressure was monitored with a manometer. To investigate the influence of arachidonic acid on the channel opening, we applied arachidonic acid (Wako, Osaka, Japan), at concentrations of 0.1 to 100 µM, to the cytosolic sides of patch membranes by perfusion of intracellular solution in inside-out patches. Stock solutions of arachidonic acid (300 mM) in ethanol were diluted in the intracellular solution before the experiments. The stock solutions were kept under nitrogen gas, stored at –25°C, and used within one week. Experiments were done at room temperature (23–26°C).

Only currents recorded from patches with over 30-G seal resistances were used for analysis. Currents were recorded on an RD-120TE data recorder (TEAC, Tokyo, Japan). For analysis of the currents, data records were digitized at 250 µs/point, after being low-pass-filtered at 1 kHz with a 4-pole Bessel filter (Nihon Kohden, Tokyo, Japan). The digitized currents were analyzed with pClamp9 software (Axon Instruments, Foster City, CA, USA). Channel activity is reported as the open probability times the number of channels in a patch (NP_o). Data are expressed as means ± SD, and n indicates the number of experiments. Statistical analysis was performed with Student’s t test. A value of p < 0.05 was considered to be statistically significant.

Total RNA Extraction and RT-PCR
For the detection of expression of TREK-1 K⁺ channel, total RNA was extracted from the cultured PDL fibroblasts with the use of TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA), and was then subjected to oligo dT primed first-strand cDNA synthesis with SuperScript II Reverse Transcriptase (Invitrogen). PCR amplification was performed from the RT mixture corresponding to 50 ng of total RNA, with AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) and gene-specific primers. PCR primers for human TREK-1, TREK-2 (Lesage et al., 2000), and GAPDH (Nishikawa, 2002) were used. The primers of TREK-1 (sense primer 5'-AACAACTATTGGATTTGGTG-3', antisense primer 5'-GGCTATTTGATGTTCTCAAT-3', corresponding to positions 761–780 and 1233-1252, respectively) were designed with the use of Primer 3 software based on the TREK-1 mRNA sequence (GenBank AF171068). The amplification was carried out for 33 cycles of 30 sec at 95°C and 1 min at 62°C, followed by 10 min at 72°C. PCR products were separated by electrophoresis on 1.2% agarose gel and visualized by ethidium bromide staining under UV light.

Immunocytochemistry
PDL fibroblasts were fixed with methanol for 10 min and acetone for 1 min at –20°C. After being rinsed with PBS, the cells were incubated in goat anti-human TREK-1 polyclonal antibodies (sc-11556; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; dilution 1:200) at 4°C for 24 hrs. After being washed, the cells were incubated in 5 µg/mL FITC anti-goat IgG (Alexa Fluor 488, A11078; Molecular Probes, Inc., Eugene, OR, USA), washed again, mounted in glycerol, and observed by fluorescence microscopy. As a negative control, goat IgG was substituted for the anti-human TREK-1 antibody.

	RESULTS

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The activity of the TREK-1 K⁺ channel in cultured human PDL fibroblasts was low in both cell-attached and inside-out patches (NP_o, about 0.01). To obtain the single-channel conductance and to determine the ion selectivity of the channel, we recorded single-channel currents in inside-out patches, with the addition of 3 µM arachidonic acid to the bath solution to enhance the channel activity. The channel currents were highly variable (representative current traces at various membrane voltages shown in Fig. 1A). The current-voltage (I-V) relationships from inside-out patches in symmetrical high-K⁺ solutions could be fitted to a linear regression line, showing that the single-channel slope conductance of the TREK-1 K⁺ channel was 104 pS (solid line in Fig. 1B). The I-V relationships obtained from the experiments in which the bath solution (dotted line) or the pipette solution (dashed line) was replaced by the high-Na⁺ solution reveal that the ion selectivity of the channel is for K⁺.

View larger version (38K): [in this window] [in a new window]   Figure 1. Single-channel currents and current-voltage (I-V) relationships of TREK-1 K+ channels obtained from human PDL fibroblasts. (A) Current traces from an inside-out patch. Voltages on the left are negative pipette potentials, i.e., in inside-out mode, the voltage values show absolute potentials of the patch membrane (Vm). Horizontal lines to the right of each trace indicate the closed state. Inward currents are downward. Both pipette solution and bath solutions were the high-K+ solution. The bath solution contained 3 µM arachidonic acid. (B) I-V relationships obtained from inside-out patches. Currents were recorded from patches given a serial voltage pulse every 20 mV between -100 mV and 100 mV. Negative current is inward. Datapoints show means ± SD. The solid line indicates the I-V relationship obtained from inside-out patches in symmetrical high-K+ solutions (n = 11). The I-V relationship can be fitted to a regression line, current = 0.104 Vm + 0.022 (R2 = 0.999), showing that the single-channel slope conductance of the TREK-1 K+ channel is 104 pS. The dotted line indicates the I-V relationship obtained from several patches from among the same patches, the bath solution of which was exchanged to the high-Na+ solution by perfusion, and currents were recorded again for the serial voltage pulses (n = 8). The I-V relationship showed a slight inward rectification (that is, the I-V relationship is not linear and binding). As the voltage rose, the currents became too small to be detected, but the currents recorded at voltages lower than the reversal potential of the potassium ion (about 83 mV) flowed inwardly. The dashed line indicates the I-V relationship obtained from patches with high-Na+ pipette solution and high-K+ bath solution (n = 9). The I-V relationship showed a slight outward rectification, and distinct inward currents could not be detected, but the currents flowed outwardly at voltages higher than the reversal potential of the potassium ion (about -83 mV). These results show that the channel is a K+-selective channel.

View larger version (29K): [in this window] [in a new window]   Figure 2. Membrane stretch induced channel opening. We elicited membrane stretch by applying suction (negative pressure). (A) Current traces under various conditions. Horizontal bars above the traces represent the durations of mechanical stress. Horizontal lines of each trace represent the closed state. Vp, pipette potential; Vm, membrane potential. (upper panel) A current trace from a cell-attached patch with high-K+ pipette solution. Vp = –20 mV and –40 mV. (middle panel) A current trace from an inside-out patch in symmetrical high-K+ solutions. Vm = –20 mV and –40 mV. (lower panel) A current trace from an inside-out patch in symmetrical high-K+ solutions whose bath solution contained 3 µM arachidonic acid. Vm = 20 mV. (B) The strength of the negative pressure to initiate the activation of channel opening. Each point is expressed as mean ± SD (n = 10). The strength was smaller in inside-out patches (without arachidonic acid, aa-free) than in cell-attached patches (cell-attached). Arachidonic acid at 3 µM (3 µM aa) significantly reduced the pressure strength required to initiate the increase in channel activity. *p < 0.001 vs. cell-attached. +p < 0.0001 vs. aa-free.

View larger version (25K): [in this window] [in a new window]   Figure 3. Arachidonic-acid-activated TREK-1 K+ channel opening. (A) Current traces from an inside-out patch with high-K+ bath solution containing arachidonic acid at various concentrations. The pipette solution was also the high-K+ solution. Vm = 20 mV. (B) Dose-dependent effects of arachidonic acid on the activity of the TREK-1 K+ channel (means ± SD, n = 8). The channel activity is expressed as the percentage of NPo at each concentration relative to NPo at 100 µM of arachidonic acid (%NPo). NPo values were obtained from current recordings in patches where the concentration of arachidonic acid in bath solution was altered between 0 µM and 100 µM by perfusion. Abscissa, common logarithmic scale. (C) %NPo when K+ channel blockers were applied to the bath solution in inside-out patches in which TREK-1 K+ channels had been activated by 10 µM arachidonic acid. Ba2+ at a rather high concentration (3 mM) decreased channel activity. Quinine and quinidine were more effective than Ba2+. Each value was obtained from five inside-out patches (mean ± SD, n = 5). *Significantly different from blocker-free state.

View larger version (67K): [in this window] [in a new window]   Figure 4. RT-PCR and immunohistochemistry of the TREK-1 K+ channel in cultured PDL fibroblasts. (A) Analysis of the expression of TREK-1 by RT-PCR. Lane 1 contains the PCR product of TREK-1. The RT-PCR product migrates in the gel to a position in good agreement with its predicted size of 492 bp. Lane 2 contains PCR-amplified product of TREK-2. Lane 3 shows RT-PCR product of GAPDH (571 bp). GAPDH was amplified to verify equal loading of RNA. Lane 4 shows the DNA molecular size standard. The results show that cultured human PDL fibroblasts do express TREK-1, but not TREK-2. (B) Immunocytochemical staining of TREK-1 K+ channels in cultured human PDL fibroblasts. PDL fibroblasts were stained with goat anti-human TREK-1 polyclonal antibody (TREK-1) or IgG as a negative control (IgG). White bar, 20 µm.

	DISCUSSION

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There have been few reports concerning ionic channels in PDL fibroblasts. However, it has been suggested that the intracellular Ca²⁺ concentration is increased by Ca²⁺ influx through Ca²⁺ -permeable ionic channels. PGE₂, substance P (Nohutcu et al., 1993), and histamine (Niisato et al., 1996) cause increased intracellular Ca²⁺ concentrations. The membrane stretch produced by cell swelling induced increased intracellular Ca²⁺ concentrations, which were blocked by EGTA or gadolinium, implying that the intracellular Ca²⁺ elevation resulted from the Ca²⁺ influxes (Bibby and McCulloch, 1994). Our preliminary work has shown that, besides the TREK-1 channel, an 8-pS Ca²⁺-permeable non-selective cation channel and a 250-pS Ca²⁺-activated BK potassium channel are present in human PDL fibroblasts (unpublished observations). TREK-1 K⁺ channels, cooperating with BK channels, may work to maintain or repolarize the fibroblast membranes, opposing the depolarizing stimulus of Ca²⁺ influx through the non-selective channels, especially evoked by membrane stretch.

TREK-1 K⁺ channel is activated by various physical and chemical stimuli, including mechanical stress and cell swelling (Patel et al., 1998), temperature (Maingret et al., 2000a), intracellular acidosis (Maingret et al., 1999), and polyunsaturated fatty acids (Maingret et al., 2000b). It has recently been reported that the TREK-1 channel plays an important role in neuroprotection during brain ischemia (Lauritzen et al., 2000; Buckler and Honoré, 2005; Caley et al., 2005)—namely, during brain ischemia, endogenous arachidonic acid is released, intracellular pH falls, and neurons swell. These pathological alterations will contribute to opening TREK-1 channels, and the resulting hyperpolarization will consequently reduce Ca²⁺ influx through voltage-gated Ca²⁺ channels. Thus, the activation of TREK-1 channels represents an important neuroprotective switch. The PDL, as the suspensory ligament for teeth, is frequently subjected to harsh environmental stimuli, including masticatory forces. TREK-1 K⁺ channels in PDL fibroblasts may play a cellular-protective role to maintain the membrane potential against environmental stimuli.

Recently, intriguing work on sensory transduction in human odontoblasts has been reported (Magloire et al., 2003). RT-PCR and in situ hybridization experiments have shown the expression of TREK-1 channels. Additionally, immunohistochemical observations have revealed that the spatial distribution of TREK-1 channels is closely related to the distribution of nerve endings. From the findings, it has been suggested that the physiological role of TREK-1 channels in odontoblasts is to generate a signal to afferent nerve terminals when the channels are activated by mechanical stress. A similar mechanism of sensory transduction may work in the PDL, since human periodontal ligaments are innervated by several kinds of sensory nerve endings (Lambrichts et al., 1992). The efflux of K⁺ from PDL fibroblasts by the activation of TREK-1 channels by mechanical stress may modify the local ionic composition of the tissue fluid in PDL to send a signal to neighboring afferent nerve terminals, or to alter the sensitivities of the nerve endings. Actually, it has been reported that the locally applied potassium chloride activates A fibers of the afferent nerve in cat PDL (Mengel et al., 1993).

	ACKNOWLEDGMENTS

 
We thank Mr. Takefumi Yamamoto of the Central Research Laboratory, Shiga University of Medical Science, for his kind technical assistance with the fluorescence microscopic studies. This work was supported by the Research Fund of Shiga University of Medical Science. A preliminary report was presented at the 80th Annual Meeting of the Physiological Society of Japan (Fukuoka, March 24–26, 2003).

Received November 18, 2004; Last revision March 21, 2006; Accepted March 22, 2006

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