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. 2002 Feb 15;22(4):1238-47.
doi: 10.1523/JNEUROSCI.22-04-01238.2002.

Mechanosensitive ion channels in cultured sensory neurons of neonatal rats

Hawon Cho  1 , Jieun Shin, Chan Young Shin, Soon-Youl Lee, Uhtaek Oh
Affiliations

Mechanosensitive ion channels in cultured sensory neurons of neonatal rats

Hawon Cho et al. J Neurosci. .

Abstract

Mechanosensitive (MS) ion channels are present in a variety of cells. However, very little is known about the ion channels that account for mechanical sensitivity in sensory neurons. We identified the two most frequently encountered but distinct types of MS channels in 1390 of 2962 membrane patches tested in cultured dorsal root ganglion neurons. The two MS channels exhibited different thresholds, thus named as low-threshold (LT) and high-threshold (HT) MS channels, and sensitivity to pressure. The two channels retained different single-channel conductances and current-voltage relationships: LT and HT channels elicited large- and small-channel conductance with outwardly rectifying and linear I-V relationships, respectively. Both LT and HT MS channels were permeable to monovalent cations and Ca2+ and were blocked by gadolinium, a blocker of MS channels. Colchicine and cytochalasin D markedly reduced the activities of the two MS channels, indicating that cytoskeletal elements support the mechanosensitivity. Both types of MS channels were found primarily in small sensory neurons with diameters of <30 microm. Furthermore, HT MS channels were sensitized by a well known inducer of mechanical hyperalgesia, prostaglandin E2, via the protein kinase A pathway. We identified two distinct types of MS channels in sensory neurons that probably give rise to the observed MS whole-cell currents and transduce mechanical stimuli to neural signals involved in somatosensation, including pain.

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Figures

Fig. 1.
Fig. 1.
Activation of MS whole-cell currents by pressure. A,Top trace, Whole-cell currents of a cultured sensory neuron activated by pressure.Bottom trace, Positive pressures applied to the patch pipette. B, A summary of MS whole-cell currents in sensory neurons. Because of the fragile nature of whole cells, positive pressures of <20 mmHg were applied to whole cells. Membrane potential was held at −60 mV. **p < 0.01 compared with the mean of whole-cell currents activated by 5 mmHg. Numbers inparentheses represent the number of experiments.C, Current–voltage relationship of the whole-cell current activated by pressure. Voltage ramps (bottom trace) ranging from −100 to +100 mV with 300 msec duration were applied before and during pressure application. Current elicited before pressure (solid line), during pressure application (dotted line), and difference current (dashedline).
Fig. 2.
Fig. 2.
Single-channel currents activated by negative pressures applied to a cell-attached patch. A, This channel was classified as an LT MS channel because the channel was activated by a relatively low pressure.Ehold = −60 mV. Right,An amplitude histogram of the single-channel currents. The mean amplitudes of LT MS channels were best fitted by a Gaussian distribution. B, Another type of single-channel currents activated only by negative pressures greater than −80 mmHg. The channel was classified as an HT MS channel because of its high pressure threshold. The cell-attached patch contained two HT channels.Right, An amplitude histogram of the single-channel currents. C, Pressure-response relationships of the LT (filled circle;n = 9) and HT (filled triangle;n = 9) MS channels. Relative channel activity (NPo/NPmax) at each pressure was fitted to the Boltzmann distribution described in Results. Bars represent SEM.
Fig. 3.
Fig. 3.
Current–voltage relationships and kinetics of LT and HT MS channels. A, Traces in the expanded time scale of single-channel currents of the two MS channels held at different membrane potentials. Suction pressures of −60 and −80 mmHg were delivered to pipettes of inside-out patches to activate the LT and HT MS channels, respectively. B, Current–voltage relationships of single-channel currents. LT, Filled circle (n = 13); HT, filled square (n = 11). To obtain the current–voltage relationship of LT channels, each data point was fitted to a single exponential equation. Bars represent SEM. The error bars are so small that they are included by the circlesor squares. C, Open and closed time histograms of LT and HT MS channels. LT and HT channels were activated by −80 and −100 mmHg, respectively. The histograms of open and closed time durations of the two channels were best fitted by two exponential functions.
Fig. 4.
Fig. 4.
Ion selectivity of LT and HT channels.A, The N-methyl-d-glucamine substitution. No outward channel currents were observed for the two types of MS channels when the 140 mm Na+bath solution was replaced by a solution containing 140 mmN-methyl-d-glucamine (n= 3 for each MS channel). B, C, Cation selectivity of LT (B) and HT (C) MS channels. Current–voltage relationships of each type of MS channels were obtained after the symmetrical 140 mm Na+ bath solution of inside-out patches was replaced by solutions containing equimolar K+, Cs+, or Li+. The I–V relationships of the MS channels in the symmetrical 140 mm Na+salt condition are identical to those shown in Figure 3.D, E, Permeability of LT (D) and HT (E) MS channels to Ca2+. Current–voltage relationships were obtained from inside-out patches that contained 100 mmCa2+ in the pipette solution and 140 mmNa+ in the bath solution.
Fig. 5.
Fig. 5.
Block of MS channels by Gd. A,B, Example traces depicting the effects of 10 μm Gd on activities of LT (A) and HT (B) MS channels in outside-out patches. The membrane potential was held at −60 mV throughout the experiments.C, D, Summaries of the effects of Gd on activities of LT (C) and HT (D) MS channels. Numbers on thebars represent the number of experiments. **p < 0.01
Fig. 6.
Fig. 6.
Inhibition of activities of the LT and HT channels by cytoskeleton disrupters, cytochalasin D, and colchicine.A, B, Example traces of the effect of cytochalasin D on activities of LT (A) and HT (B) MS channels. Negative pressures were applied to patch pipettes to activate the MS channels after forming cell-attached patches. The cell-attached patches were incubated with 10 μm cytochalasin D for ∼20 min. The negative pressures were repeated after the cytochalasin D application. C, Summaries of the effects of repeated applications of suction pressures on the pressure-activity relationships of LT (n = 4–6) and HT (n = 4–7) MS channels. Note that repeated applications of suction pressures do not change the overall responses of the MS channels to pressures. D, Summaries of the effects of 10 μm cytochalasin D on the pressure responses of LT (n = 4–6) and HT (n = 3–7) MS channels. E, Summaries of the effects of 500 μm colchicine on the pressure responses of LT (n = 4–5) and HT (n = 3–6) MS channels.
Fig. 7.
Fig. 7.
Sensitization of HT MS channel by PGE2via the protein kinase A pathway. A, Negative pressures were repeated in cell-attached patches at a holding potential of −60 mV. Before and during the second application of suction pressures, the cell-attached patches of sensory neurons were incubated with 10 μm PGE2 for ∼5 min. Right,The pressure-activity relationships of HT MS channels before (circle;n = 6–14) and after PGE2 (triangle;n = 6–14) treatment. Channel activity (NPo) at each pressure was fitted to the Boltzmann distribution described in the text. Bars represent SEM.B, Block of the PGE2-induced sensitization of HT channels by H-89, an inhibitor of protein kinase A. H-89 (10 μm) was incubated before and during PGE2application. C, A summary of the effect of coapplication of H-89 and PGE2 on the pressure-response relationship of HT MS channels. Control (circle;n = 5–10), after H-89 treatment (triangle;n = 5–10). D, A summary of the effect of the application of dibutyryl cAMP (dbcAMP), a soluble cAMP analog, on the pressure-response relationship of HT MS channels. Control (circle;n = 5–10), after dbcAMP treatment (triangle; n = 5–10).
Fig. 8.
Fig. 8.
Cell-size proportions of cultured sensory neurons eliciting each type of MS channel or whole-cell currents in cultured DRG neurons.
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