Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons

Abstract

Low voltage-activated (LVA) T-type Ca(2+) (I(Ca)T) and NaN/Nav1.9 currents regulate DRG neurons by setting the threshold for the action potential. Although alterations in these channels have been implicated in a variety of pathological pain states, their roles in processing sensory information remain poorly understood. Here, we carried out a detailed characterization of LVA currents in DRG neurons by using a method for better separation of NaN/Nav1.9 and I(Ca)T currents. NaN/Nav1.9 was inhibited by inorganic I(Ca) blockers as follows (IC(50), microM): La(3+) (46) > Cd(2+) (233) > Ni(2+) (892) and by mibefradil, a non-dihydropyridine I(Ca)T antagonist. Amiloride, however, a preferential Cav3.2 channel blocker, had no effects on NaN/Nav1.9 current. Using these discriminative tools, we showed that NaN/Nav1.9, Cav3.2, and amiloride- and Ni(2+)-resistant I(Ca)T (AR-I(Ca)T) contribute differentially to LVA currents in distinct sensory cell populations. NaN/Nav1.9 carried LVA currents into type-I (CI) and type-II (CII) small nociceptors and medium-Adelta-like nociceptive cells but not in low-threshold mechanoreceptors, including putative Down-hair (D-hair) and Aalpha/beta cells. Cav3.2 predominated in CII-nociceptors and in putative D-hair cells. AR-I(Ca)T was restricted to CII-nociceptors, putative D-hair cells, and Aalpha/beta-like cells. These cell types distinguished by their current-signature displayed different types of mechanosensitive channels. CI- and CII-nociceptors displayed amiloride-sensitive high-threshold mechanical currents with slow or no adaptation, respectively. Putative D-hair and Aalpha/beta-like cells had low-threshold mechanical currents, which were distinguished by their adapting kinetics and sensitivity to amiloride. Thus, subspecialized DRG cells express specific combinations of LVA and mechanosensitive channels, which are likely to play a key role in shaping responses of DRG neurons transmitting different sensory modalities.

Figure 1.

Heterogeneity of low-threshold inward currents in DRG neurons. (A) Families of current traces elicited in small- (a–c) and medium- (d) diameter DRG neurons by 100-ms depolarizations. Currents were evoked by stepping from −80 to −45 mV in 2.5- or 5-mV increments from a holding potential of −100 mV. Note the difference in inactivation kinetics of these LVA currents. Membrane capacitance is indicated for each cell. (B) Block of mixed LVA current by increasing concentrations of Cd²⁺ (0–1000 μM, as indicated) in a small DRG neuron (27 pF). The bottom panel shows peak-normalized difference currents determined as indicated. (C) Cadmium block of mixed LVA currents in small-sized DRG neurons (15–29 pF, n = 6) determined isochronally 20 or 95 ms after the onset of the test pulse (inset). Data were obtained with a 100-ms test pulse to −60 mV from a holding potential of −100 mV once every 5 s and normalized to current amplitude obtained before application of drug. Data points were plotted against [Cd²⁺] in semilogarithmic scale and best fit to single site binding curves (solid and dashed lines). IC₅₀ ranged from 13 to 200 μM (n_H = 0.8–1.56) and from 149 to 381 μM (n_H = 1.05–1.2) when determined at t = 20 and t = 95 ms, respectively. Mean IC₅₀ values obtained at t = 20 and t = 95 ms were 44 ± 11 and 240 ± 35 μM, which were significantly different (P < 0.005; two-tailed unpaired t test).

Figure 2.

Amiloride blocks low-threshold T-type Ca²⁺ current but spares NaN/Nav1.9 current. (A) Inhibition of LVA currents by amiloride (1 mM) in a small DRG neuron (34 pF, a) and in a medium-diameter D-hair cell (50 pF, b). Currents were evoked by a depolarizing step to −55 mV from a holding of −100 mV and amiloride inhibition is shown at steady state. (A, c) Superimposed amiloride-sensitive LVA currents (difference currents) obtained in the corresponding small- and medium-diameter DRG neurons as indicated. Traces are scaled for comparison. (B) LVA currents evoked by a double-pulse voltage protocol in the absence and presence of 3 mM amiloride in a small DRG neuron (23 pF). The voltage protocol consisted of two 100-ms depolarizing steps to −50 mV, separated by a 4-ms interpulse to −100 mV, which was short enough to prevent repriming of T-type Ca²⁺ channels. (C) Amplitude of LVA currents plotted as a function of time for the corresponding cell shown in B. The horizontal bars indicate the time and duration of application of amiloride. The DRG neuron was stimulated every 3 s by the use of the double-pulse protocol as in B.

Figure 3.

Mibefradil block of NaN/Nav1.9 and SNS/Nav1.8 currents in small DRG neurons. (A) Inhibition of normalized NaN/Nav1.9 current by mibefradil (5 μM) in small DRG neurons. The cells were held at −100 mV and depolarized to −55 mV at 0.2 (•) or 0.5 Hz (◯). Smooth curves show single exponential fits with time constants as indicated. Insert shows mibefradil inhibition of NaN/Nav1.9 current evoked at 0.5 Hz; for clarity's sake, only 1 trace every 10 s is shown. Mean time constants for mibefradil block were 49 ± 6 and 112 ± 7 s at 0.5 and 0.2 Hz, respectively (n = 6; P < 0.05). (B) Concentration–inhibition curve for mibefradil in small DRG neurons (18–27 pF). Mibefradil was cumulatively applied at increasing concentrations (1–30 μM) for the time necessary to approach equilibrium at 1 Hz. Hill equation was used to fit data and yielded an IC₅₀ value of 5.15 ± 0.5 μM (n_H = 1.2). Each data point is the mean ± SEM of 11 observations. The insert shows superimposed NaN/Nav1.9 current in the absence or presence of increasing concentrations of mibefradil (3–30 μM). (C) Inhibition of SNS/Nav1.8 current by 10 μM mibefradil in a small DRG neuron (29 pF) in which SNS/Nav1.8 predominates. Currents were evoked by depolarizing voltage steps to 0 mV from a holding potential of −100 mV once every 2 s (0.5 Hz). For clarity, only one trace every 10 s is shown. Inset, expanded time scale. (D) Peak SNS/Nav1.8 current was plotted against time for the corresponding cell in C. All experiments were made in the presence of amiloride (1 mM).

Figure 4.

Cadmium block of NaN/Nav1.9 in small DRG neurons. (A) Typical response to increasing concentrations of CdCl₂ in a small DRG neuron (26 pF, a) and in a medium-diameter D-hair cell (40 pF, b). Test pulses to −60 mV from a holding potential of −100 mV were delivered every 5 s. Note that amiloride (1 mM) was present throughout in A (a) in order to suppress T-type Ca²⁺ currents. (B) Dose–response analysis of Cd²⁺ block of NaN/Nav1.9 in small DRG neurons (◯) and LVA currents (carried primarily by I_CaT) in medium-sized D-hair cells (•). Data were obtained with a 100-ms test pulse to −60 mV from a holding potential of −100 mV once every 5 s and normalized to peak current amplitudes measured before application of CdCl₂ (inset). Solid lines are the best least-square fits to single binding site equation. Calculated IC₅₀ values are 233 ± 5 μM (n_H = 1.1) for NaN/Nav1.9 and 28 ± 2 μM (n_H = 0.81) for I_CaT. 8–11 cells per point. (C) Same cell as in A (a). Peak currents were plotted as a function of potential in control condition (0 μM Cd²⁺ + 1 mM amiloride) and in the presence of 30, 100, 300, and 1,000 μM Cd²⁺, added cumulatively. Smooth curves represent modified Boltzmann fits, giving V_1/2 and slope factors of −59.5 and 4.5 mV (◯), −58.8 and 4.6 mV (•), −56.6 and 4.4 mV (▴), −53.1 and 4.6 mV (▾), and −49 and 6.3 mV (♦). The dashed line represents the data obtained in the presence of 1,000 μM Cd²⁺ normalized to the maximum peak current. Top panel, the percentage block by 100 and 1,000 μM Cd²⁺ was calculated and plotted for each potential.

Figure 5.

Block of NaN/Nav1.9 in small DRG neurons by lanthanum and nickel. (A and B) Superimposed are NaN/Nav1.9 currents evoked by depolarizing voltage steps to −55 or −50 mV from a holding potential of −100 mV in the presence of increasing concentrations of La³⁺ (3–3,000 μM) (A) or Ni²⁺ (100–3,000 μM) (B). (C and D) Peak NaN/Nav1.9 currents plotted against time in the presence of increasing concentrations of La³⁺ (C) and Ni²⁺ (D). The horizontal bars indicate the time and duration of the drug application. (E and F) Cumulative concentration–inhibition curves for La³⁺ (E) and Ni²⁺ (F). Hill equation was used to fit data points. IC₅₀ values were 45.8 ± 3 μM (n_H = 0.96) for La³⁺ and 892 ± 8 μM (n_H = 0.91) for Ni²⁺. Each data point is the mean ± SEM of 7–12 observations. All experiments were made in the presence of amiloride (1 mM).

Figure 6.

LVA current signature of C-I type nociceptive cells. (A–C and E) Same cell. (A) Families of current traces elicited in a small DRG neuron (25 pF) in the absence or presence of 1 mM amiloride. Currents were evoked by 100-ms depolarizations by stepping from −80 to −10 mV in 5-mV increments from a holding potential of −100 mV. Note the marked demarcation between low- and high-threshold currents. Cluster analysis placed 31 small cells into this category. (B) LVA currents evoked at −50 mV in control conditions and after sequential application of amiloride (1 mM) and amiloride-containing low Na⁺ solution (2 mM, Low Na⁺). The right panels show difference currents, isolating NaN/Nav1.9- and Ca²⁺-components of the total LVA current. (C) Peak current–voltage relationships for the corresponding neuron illustrated in A, in the absence (Control) or presence of amiloride (1 mM) and after bath application of amiloride-containing low Na⁺ solution (Low Na⁺). (D) Relative conductances (G/G_max) of the amiloride-sensitive I_CaT (◯) and NaN/Nav1.9 (▵) were plotted against membrane potential and fitted to single Boltzmann functions. The inserts show difference currents isolating the amiloride-sensitive I_CaT and NaN/Nav1.9. Half-activation voltages and slope factors were −65 ± 1.5 and −58.5 ± 1 mV and 4 ± 0.5 and 5.5 ± 0.6 mV, respectively. Note that NaN/Nav1.9 currents were isolated by isoosmotically substituting external Na⁺. Bars, 100 pA. Each data point is the mean ± SEM of 11–14 cells. (E) Representative inward current evoked by the application of 1 μM capsaicin at the end of the experiment. 82% of these cells responded to exposure to capsaicin with a mean current of −710 ± 25 pA.

Figure 7.

LVA current signature of C-II type nociceptive cells. (A–C, E, and G) Same cell. (A) Families of current traces elicited in a small DRG neuron (16 pF) in the absence or presence of 1 mM amiloride (A, a) and in amiloride-containing low Na⁺ solution (A, b). Currents were evoked by 100-ms depolarizations as indicated. Cluster analysis placed 27 small cells into this category. (B) Families of amiloride-sensitive I_CaT derived from difference currents in A (a). Voltage protocol as in A (b). (C) Superimposed LVA currents evoked at −50 mV in control conditions (1) and after sequential application of amiloride (1 mM, 2), amiloride-containing low Na⁺ solution (Low Na⁺, 3), and 10 μM La³⁺ in amiloride-containing low Na⁺ solution (4). The bottom panel shows superimposed difference currents determined as indicated, isolating amiloride-sensitive I_CaT (1–2), NaN/Nav1.9 (2–3), and amiloride-resistant I_CaT (3–4, red trace). Note the moderate inactivation and slow tail currents of the amiloride-resistant I_CaT. (D) Current traces of the amiloride-resistant I_CaT showing relationships between test pulse duration and tail current amplitude and kinetics. Test pulses were evoked by 2.5-ms depolarization to −50 mV and the pulse duration was lengthened by 2.5 ms between each sweep. Red and blue traces illustrate currents evoked by 22.5- and 100-ms step duration. (E) Peak current–voltage relationships in the absence (Control) or presence of amiloride (1 mM) and after bath application of amiloride-containing low Na⁺ solution in the absence (Low Na⁺) or presence of La³⁺ (10 μM). (F) The relative conductances (G/G_max) of the amiloride-sensitive and amiloride-resistant I_CaT were plotted against membrane potential and fitted to single Boltzmann functions. V_1/2 and slope factors were −65 ± 0.8 and −60 ± 1.2 mV and 4.4 ± 0.3 and 5.5 ± 0.5 mV, for amiloride-sensitive and amiloride-resistant I_CaT, respectively. Each data point is the mean ± SEM of 9–11 cells. (G) Inward current evoked by the application of 1 μM capsaicin at the end of the experiment. 60% of these cells responded to exposure to capsaicin with a mean current of −245 ± 15 pA.

Figure 8.

LVA current signature of medium nociceptive cells. (A and B) Same cell. (A) Families of LVA current traces elicited in a medium-sized DRG neuron (42 pF) in the absence or presence of 1 mM amiloride and in amiloride-containing low Na⁺ solution. Currents were evoked by 100-ms depolarizations by stepping from −90 to −50 mV in 5-mV increments from a holding potential of −100 mV. Cluster analysis placed 37 medium-sized DRG neurons into this category. (B) Peak current–voltage relationships (B, a) in the absence (Control, ◯) or presence of amiloride (1 mM, •) and after bath application of amiloride-containing low Na⁺ solution (Low Na⁺, ⋄). Inset, families of amiloride-sensitive I_CaT (difference currents). Voltage protocol as in A. (B, b) Superimposed currents elicited by a 10-s ramp depolarization from −100 to +20 mV (rising rate 12 mV s⁻¹) in the presence of amiloride (1 mM) before (control) and after holding the cell at −60 mV for 5 s in order to promote slow inactivation of NaN/Nav1.9 (Slow inactivated). Leak currents were not subtracted in this recording. (C, D, and F) Same cell. (C) Families of NaN/Nav1.9 currents evoked by 500-ms depolarizations in a medium-sized DRG neuron (38 pF) in the presence of amiloride (1 mM). Note the slowly developing inactivation of the NaN/Nav1.9 current at −50 mV (τ = 232 ms; 219 ± 7 ms, n = 12). (D) Superimposed NaN/Nav1.9 currents evoked at −50 mV showing relationships between test pulse duration and tail current amplitude and kinetics. The voltage pulse duration was varied from 4 to 104 ms by 10-ms increments. Note the rapid deactivation time course of NaN/Nav1.9. Time constants obtained from fitting single exponentials to data points are indicated (filtering frequency, 5 kHz). 1 mM amiloride throughout. (E) The relative conductances (G/G_max) of NaN/Nav1.9 (▵) and amiloride-sensitive I_CaT (◯) were plotted against membrane potential and fitted to single Boltzmann functions. V_1/2 and slope factors were −58 ± 2 and −65 ± 1.2 mV and 5.3 ± 0.2 and 4.1 ± 0.2 mV, for NaN/Nav1.9 and amiloride-sensitive I_CaT, respectively. Each data point is the mean ± SEM of 7–10 cells. (F) Inward current evoked by the application of 1 μM capsaicin at the end of the experiment. 75% of these cells responded to exposure to capsaicin with a mean current of −4150 ± 45 pA.

Figure 9.

Nociceptors display two types of high-threshold mechanically activated cation currents. (A–C) Representative voltage step–evoked currents and mechanically gated inward currents observed in C-I type (25–40 pF, A), C-II type (14–28 pF, B) and Aδ-like (35–70 pF, C) nociceptors. Cells were first subjected to 100-ms step depolarizations from a holding potential of −100 mV (as indicated) and then subjected to suprathreshold mechanical stimuli (A and B) or to a series of mechanical steps in 2-μm increments (C). Note that the cell (25 pF) clustering in C-I type cells was sensitive to capsaicin (not depicted) and displayed slowly adapting MA currents (A) while the C-II type cell (17 pF) was insensitive to capsaicin (not depicted) and had MA currents that failed to adapt during the entire length of the stimulus (B). The minimum distance travelled by the probe to evoke a response in A and B was 13.5 and 13.1 μm, respectively. (C) The cell (41 pF) clustering in the Aδ-like cells was found to be unresponsive to mechanical stimuli. Sweeps were applied at 15-s intervals in C. The velocity of the probe was 200 μm s⁻¹ during the ramp segments. (D–F) Same cell as in B. (D) Relationship between pressure strength and the induced mechanical inward current. Sweeps were applied at 15-s intervals but were shown concatenated for clarity's sake. Encoding of the intensity of the stimulus was demonstrated by the graded responses to varying mechanical stimulus applied through the glass probe. Note that amplitude of the MA current saturated as higher pressure was applied. (E) The cell was subjected to dual voltage–mechanical protocol consisting of a 100-ms voltage step from −100 to −60 mV followed by a suprathreshold mechanical stimulus. Amiloride (1 mM) blocked the MA current by ∼80%. (F) Current–voltage relationships of MA current before (◯) and after (•) application of 1 mM amiloride. Reversal membrane potential was −5 mV. Inset, cation currents evoked by a 20-μm ramp stimulus at holding potentials ranging from −80 to +40 mV. Bars, 100 pA.

Figure 10.

Medium-sized D-hair cells express low-threshold, rapidly adapting MA currents and two pharmacologically distinct I_CaT. (A–D) Same cell. (A) A medium-sized D-hair cell (52 pF) was subjected to dual voltage–mechanical protocol consisting of a 100-ms voltage step from −100 to −50 mV followed by a series of incrementing (1 μm) mechanical stimuli. Sweeps were applied at 15-s intervals to allow MA currents to recover fully. D-hair cells are identifiable by the presence of large I_CaT. The minimum distance travelled by the probe to evoke a response was 7.5 μm, thus this cell was classified as low-threshold mechanoreceptor. Note that at mechanical threshold, a transient, rapidly adapting inward current was evoked while suprathreshold stimuli evoked a current with both transient and sustained components. The velocity of the probe was 200 μm s⁻¹ during the ramp segments. (B) Both I_CaT and MA currents were inhibited by amiloride (1 mM). (C) Families of LVA currents evoked in the D-hair cell by 100-ms depolarizations from −80 to −30 mV in 5-mV increments in control, in the presence of amiloride (1 mM) and after bath application of amiloride-containing low Na⁺ solution. (D) Peak I-V relationships in the absence (Control, ◯) or presence of amiloride (1 mM, •) and after bath application of amiloride-containing low Na⁺ solution in the absence (Low Na⁺, ⋄) or presence of La³⁺ (10 μM, ♦). Note that in this cell, as in most D-hair cells, a small SNS/Nav1.8-like current is seen (indicated by the arrow; see also Fig. 13). (E) The relative conductances (G/G_max) of the amiloride-sensitive (⋄) and amiloride-resistant (◯) I_CaT were plotted against membrane potential and fitted to Boltzmann functions. V_1/2 and slope factors were −65 ± 1.5 and −63 ± 1.5 mV and 4.7 ± 0.2 and 5 ± 0.1 mV, respectively. Each data point is the mean ± SEM of 7–14 cells. Insets, amplitude of the amiloride-resistant I_CaT was estimated as the La³⁺-sensitive component of LVA currents determined in the amiloride-containing low Na⁺ solution, whereas the amiloride-sensitive I_CaT is obtained by difference currents derived from experiments as in C. This type of cells was found to be unresponsive to capsaicin.

Figure 11.

Large-diameter DRG cells lacking NaN/Nav1.9 express low-threshold intermediately adapting MA currents and two types of I_CaT. (A–E) Same cell. (A) Families of current traces elicited in a large DRG neuron (87 pF) in the absence (Control) or presence of 1 mM amiloride and in amiloride-containing low Na⁺ solution. Currents were evoked by 100-ms depolarizations by stepping from −80 to −35 mV in 5-mV increments from a holding potential of −100 mV. Cluster analysis placed 18 large-sized DRG neurons into this category. Bottom traces show difference currents isolating the amiloride-sensitive I_CaT (steps from −80 to −50 mV) and the TTX-R SNS-like current (steps from −80 to −35 mV). Note that in addition to amiloride-resistant I_CaT, these large cells also expressed large high-threshold I_Ca (arrow), which was partially blocked by our fluoride-containing pipette solution. (B) Peak I-V relationships in the absence (Control, ◯) or presence of amiloride (1 mM, •) and after bath application of amiloride-containing low Na⁺ solution in the absence (⋄) or presence of La³⁺ (10 μM, ♦). Inset, block of the amiloride-resistant I_CaT by 10 μM La³⁺ in the presence of low Na⁺ external solution. (C) MA currents evoked by a series of incrementing (1.5 μm) mechanical stimuli at a holding potential of −80 mV. The minimum distance travelled by the probe to evoke a response was ∼8 μm, thus this cell was classified as low-threshold mechanoreceptor. Note that decay kinetics of MA currents had time constants in between slowly adapting nociceptors and rapidly adapting D-hair cells. Sweeps were applied at 15-s intervals; probe velocity, 200 μm s⁻¹. (D) Amplitude (bottom, ◯) and decay kinetics (top, •) of the MA current plotted as a function of holding potential. The cell was held at V_h for at least 20 s before the mechanical stimulation (15 μm) was applied. The holding currents were subtracted for clarity. Inset, MA currents evoked by a 15-μm ramp stimulus at holding potentials ranging from −50 to +20 mV. The expanded time scale shows monoexponential fits to the current decay. (E₁) Normal external solution. (E₂) Low Na⁺ external solution ([Ca²⁺]_o = 2.5 mM). The cell was subjected to a suprathreshold mechanical stimulus (E₁) or dual mechanical–voltage protocol (E₂) consisting of a suprathreshold mechanical stimulus followed by a 100-ms voltage step to −60 mV. Note that under both conditions, amiloride (1 mM) suppressed the rapidly adapting component of the low-threshold MA current but not the more sustained component. This cell was found to be unresponsive to capsaicin.

Figure 12.

Distribution patterns of ion currents in subclassified sensory neurons. (A) Histogram illustrating current signatures of small, medium, and large DRG neurons. Cells were classified according to their size and to the pattern of NaN/Nav1.9, SNS/Nav1.8, amiloride-sensitive and amiloride-resistant I_CaT. Based on these variables, cluster analysis identified five main populations among the 162 DRG cells that could be successfully tested for the entire battery of characteristics. 82, 60, 75, 0, and 5.5% of C-I type, C-II type, Aδ-like, D-hair, and Aα/β-like cells were sensitive to capsaicin. Note that whether cells were sensitive to capsaicin or not, all cells clustering as C-I type, C-II type, and Aδ-like cells had uniform LVA current signatures. Bars represent the mean ± SEM and numbers in the parentheses denote the number of cells for each class. ***, P < 0.005. (B) Comparison between representative MA currents evoked by mechanical ramp stimuli in D-hair cells, Aα/β-like cells, and C-I and C-II type nociceptors. Note the difference in threshold and kinetics of MA currents in these four subclassified cell classes. Time constants of adapting kinetics of MA currents in C-I nociceptors, D-hair cells, and Aα/β-like cells were significantly different (P < 0.05; one-way ANOVA) and were 275 ± 14 ms, 42 ± 5 ms, and 79 ± 6 ms, respectively. (C) Inhibition (mean ± SD) of peak MA currents by 1 mM amiloride. RA, rapidly adapting; IA, intermediately adapting; SA, slowly adapting; NA, nonadapting.

Figure 13.

Current signatures of nociceptors and mechanoreceptors. Current signature patterns evoked by voltage steps (100 ms), mechanical stimulation, and drug application are shown for each cell population. The protocol waveforms are illustrated in the bottom row. Five distinct cell types were identified from small-, medium-, and large-diameter cell populations. C-I and C-II type nociceptors are distinguished by the amplitude of the amiloride-sensitive I_CaT (Cav3.2), the presence of amiloride-resistant I_CaT (putative Cav3.3), and, when detected, the properties of the MA current. The combination of large amiloride-sensitive I_CaT (Cav3.2), rapidly adapting MA currents, unresponsiveness to capsaicin, and absence of NaN/Nav1.9 is particularly important to distinguish D-hair cells from medium-sized Aδ-like cells. The low-threshold mechanoreceptors, Aα/β-like and D-hair cells, are distinguished by the kinetics of their MA currents. The approximate incidence (%) is indicated when necessary.