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Single-channel Recordings of TREK-1 K+ Channels in Periodontal Ligament Fibroblasts
A. Ohara1,*,
Y. Saeki2,
M. Nishikawa3,
Y. Yamamoto4 and
G. Yamamoto3
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
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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.
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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.
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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.
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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.
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Journal of Dental Research, Vol. 85, No. 7,
664-669 (2006)
DOI: 10.1177/154405910608500716
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