GTP-dependent run-up of Piezo2-type mechanically activated currents in rat dorsal root ganglion neurons
© Jia et al.; licensee BioMed Central Ltd. 2013
Received: 23 October 2013
Accepted: 5 December 2013
Published: 17 December 2013
Rapidly adapting mechanically activated channels (RA) are expressed in primary afferent neurons and identified as Piezo2 ion channels. We made whole-cell voltage-clamp recordings from cultured dorsal root ganglion (DRG) neurons to study RA channel regulation. RA currents showed gradual increases in current amplitude (current “run-up”) after establishing whole-cell mode when 0.33 mM GTP or 0.33 mM GTPγS was included in the patch pipette internal solution. RA current run-up was also observed in HEK293 cells that heterologously expressed Piezo2 ion channels. No significant RA current run-up was observed in DRG neurons when GTP was omitted from the patch pipette internal solution, when GTP was replaced with 0.33 mM GDP, or when recordings were made under the perforated patch-clamp recording configuration. Our findings revealed a GTP-dependent up-regulation of the function of piezo2 ion channels in DRG neurons.
The ability of animals to detect noxious mechanical stimuli and innocuous touch is essential for life. For example, by detecting noxious mechanical stimuli, one can avoid potentially harmful physical forces. Under pathological conditions such as tissue inflammation and chronic nerve injury, the responses to mechanical stimuli can be greatly enhanced resulting in mechanical allodynia, an exaggerated pain state induced by gentle touch. How mechanical responses are altered under pathological conditions remains to be understood. Several places along the somatosensory system, from peripheral afferent nerve endings to the central sites in the spinal cord and brain, can be involved in the regulation of mechanical responses. Mechanical responses usually start from mechanotransduction at primary afferent nerve endings, a biological process that converts mechanical stimuli into electrical signals. Up-regulation of mechanotransduction at primary afferent nerve endings has been proposed to be a mechanism underlying mechanical allodynia and hyperalgesia. Second messenger systems, including G-protein-coupled receptors, protein kinase A and protein kinase C have been suggested to be involved in up-regulation of mechanotransduction [1, 2]. However, mechanotransduction regulation in primary afferent neurons is only partially understood.
Mechanically activated ion channels (MA) have been recognized as the main mechanical transducers in primary afferent nerves. MA currents were first recorded by McCarter et al.  from rat dorsal root ganglion (DRG) neurons in culture by using the whole-cell patch-clamp recording technique. Subsequent studies showed that at least two types of whole-cell MA currents could be evoked from DRG neurons, rapidly adapting (RA) and slowly adapting currents (SA) . The RA currents were found to be mediated by nonselective cation channels . It was shown that MA currents were up-regulated via a transcriptional mechanism by the proinflammatory neurotrophin nerve growth factor . Activators of PKC, given in vitro and in vivo, also caused increases in MA currents and behavioral sensitization to mechanical stimulation, respectively .
The study of molecular mechanisms of mechanotransduction has recently advanced with the identification of Piezo proteins as MA channels in mammals . Piezo2, one of the two Piezo channels cloned from a mouse neuroblastoma cell line , was shown to be expressed in DRG neurons. Knockdown of Piezo2 in DRG neurons specifically reduced RA currents, suggesting that RA currents were mediated by Piezo2 channels . Interestingly, Piezo2 current amplitude was increased and inactivation slowed by bradykinin receptor beta2 activation, and the up-regulation of Piezo2 function was found to be mediated by protein kinase A and protein kinase C . In addition, a role for Piezo2 in EPAC1-dependent mechanical allodynia has been reported recently . Epac1 is a guanine nucleotide exchange factor that activates Rap, a small GTP-binding protein of the Ras family of GTPases .
While recording RA currents in cultured DRG neurons, we noticed that RA current amplitude increased over time. It has been known that some ion channels such as voltage-gated Ca2+ channels showed increased currents over time during recordings, a phenomenon that is called current run-up [9–12]. RA current run-up has not been reported previously. Therefore, we explored the RA current run-up phenomenon in DRG neurons and identified GTP as a factor involved in the RA current run-up.
Materials and methods
Adult Sprague Dawley rats (250–350 g, both genders) were used. Animal care and use conformed to National Institutes of Health guidelines for care and use of experimental animals. Experimental protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee. DRG neuron cultures were prepared as described previously . In brief, rats were deeply anesthetized with isoflurane (Henry Schein, NY) and sacrificed by decapitation. DRGs were rapidly dissected out bilaterally in Leibovitz-15 medium (Mediatech Inc. VA) and incubated for 1 hour at 37°C in minimum essential medium for suspension culture (S-MEM) (Invitrogen, Grand Island, NY) with 0.2% collagenase and 0.5% dispase and then triturated to dissociate neurons. The dissociated DRG neurons were then plated on glass coverslips pre-coated with poly-D-lysine (PDL, 12.5 μg/ml in distilled H2O) and laminin (20 μg/ml in Hank’s Buffered Salt Solution HBSS, BD bio-science), and maintained in MEM culture medium (Invitrogen) that also contained nerve growth factor (2.5 S NGF; 10 ng/ml; Roche Molecular Biochemicals, Indianapolis, IN), 5% heat-inactivated horse serum (JRH Biosciences, Lenexa, KS), uridine/5-fluoro-2′-deoxyuridine (10 μM), 8 mg/ml glucose, and 1% vitamin solution (Invitrogen). The cultures were maintained in an incubator at 37°C with a humidified atmosphere of 95% air and 5% CO2. Cells were used after culturing for 3 to 12 days.
Coverslips with cultured neurons were placed in a 0.5-ml microchamber, mounted on an Olympus IX70 inverted microscope (Olympus, USA), and continuously perfused with a normal bath solution at 2 ml/min. The normal bath contained (in mM) 145 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, pH 7.3 and osmolarity of 320 mOsm. Unless otherwise indicated, bath solution was maintained at room temperature of 23°C. For conventional whole-cell recordings, the normal patch-clamp internal recording solution contained (in mM) 70 Cs2SO4, 5 KCl, 2.4 MgCl2, 0.5 CaCl2, 5 EGTA, 10.0 HEPES, 5.0 Na2ATP, 0.33 GTP-Tris salt, pH was adjusted to 7.35 with CsOH and osmolarity was adjusted with sucrose to 320 mOsm. In some conventional whole-cell recording experiments, GTP was omitted or replaced with the same amount of GDP or GTPγS in the internal recording solution. For perforated patch-clamp recording experiments, recording electrodes were filled with internal solution that contained (in mM) 70 Cs2SO4, 5 KCl, 2.4 MgCl2, 0.5 CaCl2, 5 EGTA, 10.0 HEPES; pH was adjusted to 7.35 with CsOH and osmolarity was adjusted with sucrose to 320 mOsm; and 60 μg/ml amphotericin B was added just before recordings.
Recordings were performed on small DRG neurons with diameters ranging from 25 to 35 μm. Recording electrode resistance was 3–6 MΩ, and membrane access resistance in the whole-cell configuration was ~10 MΩ and was not compensated. Junction potential between bath and electrode solution was calculated to be 11 mV and was corrected for in the data analysis. Voltage-clamp recordings were performed with cells held at -71 mV (command voltage of -60 mV minus 11 mV for junction potential correction). Signals were recorded with an Axopatch 200B amplifier, filtered at 2 KHz and sampled at 5 kHz using pCLAMP 9.0 (Axon Instruments).
Mechanical stimulation was applied to DRG cell bodies using a heat-polished glass pipette as a probe. The tip size of the probe was approximately 4 μm in diameter. The probe was controlled by a piezo-electric device (Physik Instruments, Auburn, MA) and positioned at an angle of 45° to the surface of the dish. The tip of the probe and the recorded cell were visualized as live images on a monitor throughout the recording. The live images were captured continuously through a CCD camera that was connected to the microscope with 40x objective. The tip was positioned in such a way that a 2 μm movement did not contact the cell, 3 μm had a visible contact but little membrane movement, and a 4 μm stimulus produced an observable membrane deflection. Therefore, tip forward steps of 2 μm and 3 μm were assigned as position -1 and 0 μm, respectively; a 4 μm forward step was recorded as an initial step of 1 μm membrane displacement. The probe was moved at a speed of 0.5 μm/ms. For conventional whole-cell patch-clamp recordings, a fixed mechanical stimulation was applied immediately after breaking into whole-cell mode and then continually applied at an interval of 2 min for up to 30 min. For perforated patch-clamp recordings, a period of 20 min was allowed to achieve good perforation after gigaseal formation, and mechanical stimulation was then applied in the same manner as for conventional whole-cell patch clamp recordings. To ensure the reproducibility of membrane displacement, under visual guidance we corrected any drift just before applying mechanical stimulation in each test. In some experiments, a series of graded membrane displacement steps were applied at 1 μm increments each with 500 ms duration at the beginning and end of the recording. Since membrane displacement might affect the membrane seal and thereby potentially change the access resistance, membrane properties were continually monitored during each recording. This was achieved by applying 5 mV test pulses. Data were discarded if membrane access resistance changed during recordings.
In one set of experiments, Human Embryonic Kidney 293 (HEK293) cells that expressed mouse Piezo2 ion channels were tested. HEK293 cells were grown in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum, 50 units/ml penicillin and 50 μg/ml streptomycin. Cells were plated onto 12-mm round glass coverslips placed in 12-well plates and transfected using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. For Piezo2 overexpression experiments, 1 μg/ml of mouse Piezo2 was co-transfected with 0.3 μg /ml GFP to identify transfected cells and cells were recorded 12–24 hours later. Mechanically activated currents in Piezo2-expressing HEK293 cells were recorded under the conventional whole-cell configuration with normal patch-clamp internal recording solution contained 0.33 GTP. Mechanical stimulation was applied in the same fashion as that in the recordings of RA currents in DRG neurons.
Whole-cell recording data were analyzed using Clampfit 9 software. Data are reported as mean ± SEM. Statistical significance (P < 0.05) was assessed by Analysis of Variance (ANOVA, one way) followed by multiple comparison using Student’s t-tests with no corrections [14, 15]. Linear regression was applied to assess the trend of MA current changes over time with P < 0.05 being significantly different from the slop of zero (no change of MA currents).
Mechanotransduction at nociceptive afferent nerve endings normally has high thresholds. An up-regulation of MA channel function would reduce mechanical stimulation thresholds. This is a putative mechanism underlying mechanical hyperalgesia and allodynia. In the present study, we found that RA currents in small-sized DRG neurons were increased over time in a GTP-dependent manner. This is the first report on the role of GTP in regulating RA currents in DRG neurons. The finding may help further understanding of how Piezo2-type MA channels are regulated by intracellular signaling pathways in primary afferent neurons.
We found that RA currents in cultured DRG neurons and Piezo2-medaited RA currents in HEK293 cells increased over time (run-up) during recordings using internal solutions that contained GTP. On the other hand, we did not observe RA current run-up in DRG neurons when GTP was omitted from the internal recording solution. In most previous studies on RA currents in DRG neurons GTP was not included in the internal recording solutions during conventional whole-cell recordings [3, 5, 7], and RA current run-up was not reported by these previous studies. This seems to be consistent with our finding that RA current run-up was not observed in recordings with the internal solution in which GTP was omitted. RA currents in Piezo2-expressing HEK293 cells and DRG neurons were not reported to have run-up phenomena in two previous studies using internal recording solutions that contained 0.4-0.5 mM GTP [2, 6]. It is not clear whether these two studies neglected to mention RA current run-up or because some of their experimental conditions were different from ours. While we showed RA current run-up when conventional whole-cell recordings were performed with internal solutions that contained GTP, We did not observe RA current run-up under the perforated patch-clamp recording configuration. Similarly, in previous studies using the perforated patch-clamp recordings, RA current run-up was also not reported [1, 16]. We showed that RA current run-up did not occur when GTP was replaced with GDP, suggesting that RA current run-up was specifically dependent on GTP but not on its metabolite GDP. We found that RA current run-up remained when GTP was replaced with GTPγS, a non-hydrolysable GTP. This suggests that GTP hydrolysis is not required for the RA current run-up.
Intracellular GTP is an important second messenger involved in a number of intracellular signaling pathways. The signaling roles of GTP are largely via different types of GTPases. GTPases are usually active or ‘ON’ when they bind to GTP and inactive or ‘OFF’ when they bind to GDP. It is possible that GTP-dependent run-up of RA currents is mediated by some GTPases, such as G-proteins. Bradykinin receptor beta 2, a G-protein-coupled receptor, has recently been shown to mediate the enhancement of piezo2-type MA currents via protein kinase A and protein kinase C signaling pathways . Enhancement of MA currents by protein kinase C in DRG neurons was thought to be a result of the insertion of new MA channels into the cell membrane . In addition to G-proteins, GTP-bound small GTPases such as Ras, Rho, and Rab families have been shown to regulate ion channel function by affecting ion channel membrane trafficking, protein-protein interactions, and other regulatory mechanisms . GTP has also been known to play important roles in cytoskeleton functions such as direct involvement in microtubule dynamics , and a change of cytoskeleton function may affect membrane mechanics and thereby affect MA channel function. In summary, GTP-dependent RA current run-up shown in the present study raises several possibilities for the regulation of piezo2-type MA channels by intracellular signaling pathways.
We thank J. Strong for comments on an earlier version of this manuscript and A Patapoutian for provide mouse Piezo2 plasmid. ZJ, RI and JL contributed to experimental design, data acquisition and analysis. JGG contributed to experimental design, data analysis and interpretation. This work was supported by a NIH grant DE018661 to JGG a scholarship to ZJ from Nature Science Foundation of China (NSFC, 31000376) and Hebei Province of China (C2011206026), and a travel fellowship to RI from the Mochida Memorial Foundation for Medical and Pharmaceutical Research of Japan. The authors declare no conflict of interests.
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