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Single-channel Recordings of TREK-1 K+ Channels in Periodontal Ligament Fibroblasts
1 Department of Bioscience, Correspondence: ohara{at}belle.shiga-med.ac.jp
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
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.
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
Only currents recorded from patches with over 30-G
Total RNA Extraction and RT-PCR
Immunocytochemistry
The activity of the TREK-1 K+ channel in cultured human PDL fibroblasts was low in both cell-attached and inside-out patches (NPo, 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+.
To examine the effects of mechanical stress on the channel activity, we elicited membrane stretch by suction. Interestingly, membrane stretch induced an increase in channel activity in both cell-attached and inside-out patches (Fig. 2 ). Stronger stress induced a greater response (middle panel, Fig. 2A), and repeated applications of suction did not change the strength of negative pressure to initiate the channel activation (data not shown). The strength of the negative pressure to initiate the channel opening was significantly different between cell-attached and inside-out patches (Fig. 2B). The application of arachidonic acid to the bath solution in inside-out patches further reduced the pressure strength to activate the channel.
Application of arachidonic acid to the bath solution in inside-out patches enhanced TREK-1 K+ channel activity (Figs. 3A, 3B ). Arachidonic acid at 0.3 µM induced a detectable activation of the channel. Arachidonic acid at about 4 µM caused half-maximal stimulation, and 10 µM arachidonic acid induced almost maximal response (%NPo = 86.0 ± 5.0, p = 0.072 vs. 100 µM arachidonic acid). Several K+ channel blockers were applied to patches in the presence of 10 µM arachidonic acid. Tetraethylammonium (1–5 mM), 4-aminopyridine (1–3 mM), Cs+ (1–5 mM), and iberiotoxin (100 nM) had no effects on the K+ channel opening (data not shown). But Ba2+ at a rather high concentration (3 mM) significantly decreased the channel activity, and quinine and quinidine more strongly blocked the channel opening (Fig. 3C).
To track the presence of the TREK-1 K+ channel in the human PDL fibroblasts, we conducted RT-PCR and immunocytochemical experiments (Fig. 4 ). RT-PCR analysis clearly showed the expression of TREK-1 mRNA, but not of TREK-2 (Fig. 4A). In immunohistochemical experiments, PDL fibroblasts were stained with goat anti-human TREK-1 poly-clonal antibody; some were left unstained as a negative control (Fig. 4B).
In this study, we first recorded highly variable single-channel currents of 104 pS TREK-1 K+ channels in cultured human PDL fibroblasts by patch-clamp techniques. The channel activity was low, but it was appreciably enhanced by the application of mechanical stress on the patch membrane, and by the addition of arachidonic acid to the intracellular solution. Among the blockers for K+ channels, quinine and quinidine most strongly inhibited the channel activities. These channel properties are consistent with the properties of TREK-1 K+ channels reported in previous works (Patel et al., 1998; Maingret et al., 2000b). RT-PCR and immunocytochemical experiments showed the expression of TREK-1 K+ channel. The patch-clamp results suggest that TREK-1 K+ channels may function to hyperpolarize the PDL fibroblast membranes when masticatory stress is placed on teeth. 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
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 for publication November 18, 2004. Revision received March 21, 2006. Accepted for publication March 22, 2006.
Journal of Dental Research, Vol. 85, No. 7,
664-669 (2006)
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ABSTRACT
INTRODUCTION
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). 
fibers of the afferent nerve in cat PDL (