Ca2+ permeability of nicotinic acetylcholine receptors from rat dorsal root ganglion neurones

Abstract

Ca2+ entry through neuronal nicotinic ACh receptors (nAChRs) modulates many biological processes in nervous tissue. In order to study the functional role of nAChRs in peripheral sensory signalling, we measured their Ca2+ permeability in rat dorsal root ganglion (DRG) neurones, and analysed the effects of nAChR-mediated Ca2+ influx on the function of the vanilloid receptor TRPV1. The fractional Ca2+ current (P(f), i.e. the percentage of current carried by Ca2+ ions) flowing through nAChR channels was measured by Ca2+ imaging fluorescence microscopy in combination with the patch-clamp technique. Functional nAChRs were expressed in a subset of adult DRG neurones (about 24% of the cells), typically with small to medium size as measured by their capacitance (40 +/- 3 pF). In most cells, ACh evoked slowly desensitizing currents, insensitive to methyllycaconitine (MLA, 10 nm), a potent antagonist of homomeric nAChRs. Fast decaying currents, probably mediated by alpha7*-nAChRs (i.e. native alpha7-containing nAChRs), were observed in 15% of ACh-responsive cells, in which slowly decaying currents, mediated by heteromeric nAChRs, were simultaneously present. The nAChRs of adult DRG neurones exhibited a P(f) value of 2.2 +/- 0.6% in the presence of MLA and 1.9 +/- 0.6% (P > 0.1) in the absence of MLA, indicating that homomeric MLA-sensitive nAChRs do not contribute to Ca2+ entry into adult DRG neurones. Conversely, 10% of neonatal DRG neurones showed ACh-evoked currents completely blocked by MLA. In these neurones, nAChRs showed a larger P(f) value (9.5 +/- 1.5%), indicating the expression of bona fide alpha7*-nAChRs. Finally, we report that Ca2+ influx through nAChRs in adult DRG neurones negatively modulated the TRPV1-mediated responses, representing a possible mechanism underlying the analgesic properties of nicotinic agonists on sensory neurones.

Figure 1. Whole-cell current through nicotinic acetylcholine receptors (nAChRs) in adult dorsal root ganglion neurones

A, samples of ACh- and nicotine-evoked whole-cell currents in two different voltage-clamped dorsal root ganglion (DRG) cells. Horizontal bars represent the drug applications at indicated concentrations. Superimposed traces represent single exponential functions best fitting the data, with τ values of 0.57 s (left) and 0.42 s (right). B, whole-cell currents elicited by ACh (100 μm) in another DRG neurone in the absence or presence of methyllycaconitine (MLA; 10 nm), as indicated. Superimposed traces represent single exponential functions best fitting the data, with a τ value of 0.77 s for both left and right traces. C, whole-cell currents elicited by the indicated agonists and upon 5OH-indole treatment in a neurone different from those in A and B.

Figure 2. Ca²⁺ permeability of MLA-insensitive nAChRs expressed in adult DRG neurones

A, samples of Ca²⁺ transients elicited by either ACh or nicotine at indicated concentrations on two different adult DRG neurones. B, simultaneous recordings of the whole-cell current (top) and fluorescent transient (bottom) elicited by ACh in an adult DRG neurone equilibrated in standard medium in the presence of MLA, as indicated. Current traces and fluorescence signals are aligned and share the same time scale. Note the decrease of ΔF/F indicating the rise of [Ca²⁺]_i. C, simultaneous recordings of the whole-cell current (top) and fluorescent transient (bottom) elicited by depolarization (from −70 mV to 0 mV, as indicated) in a DRG neurone equilibrated in a medium containing only Ca²⁺ as permeant ion. D, linear relationships between ΔF/F and Q obtained from the cells shown in B and C. The P_f value, calculated by normalizing the slope obtained in B (standard medium) to the slope obtained in C (Ca²⁺ medium), is 2.5%.

Figure 3. Ca²⁺ permeability of α7*-nAChRs expressed in neonatal DRG neurones

A, whole-cell currents elicited by ACh or nicotine at indicated concentration in a single cell, with or without MLA (as indicated). B, whole-cell currents elicited by nicotine in a single neonatal DRG neurone, blocked by MLA, and enhanced by 5OH-indole, at indicated concentrations. C, fluorescence variation recorded simultaneously with the bottom whole-cell current shown in B, in the presence of 5OH-indole (1 mm). D, linear relationship between ΔF/F (i.e. Ca²⁺-dependent fluorescence variation) and Q (total electric charge) obtained from the recording shown in B and C. Resulting F/Q ratio and P_f values: 0.341 nC⁻¹ and 8.4%, respectively.

Figure 4. Effects of acute stimulation of nAChRs on the capsaicin-induced whole-cell currents in adult DRG neurones

A, simultaneous recordings of whole-cell currents (bottom) and of [Ca²⁺]_i changes (top) in a single DRG neurone. Capsaicin pulses (0.5 μm, 0.5 s) were applied every 20 s in a Ca²⁺-free medium (as indicated). Note the prolonged nicotine application (500 μm, 15 s) in the presence of normal external Ca²⁺, inducing inward current (grey line) and Ca²⁺ entry, and modifying the subsequent capsaicin-evoked currents. B, capsaicin-induced currents recorded before and after nicotine application. Current amplitudes were normalized to allow a better comparison of desensitization kinetics. Superimposed lines represent single exponential functions best fitting the data, with τ values of 3.70 s (before nicotine) and 2.69 s (after nicotine). Asterisks indicate the corresponding currents in A and B. C, time course of normalized τ of capsaicin-induced currents before and after a 15 s nicotine application (horizontal line; mean τ of the current preceding nicotine application, 3.0 ± 1.0 s). Note absence of τ recovery after nicotine washout. D, time course of normalized amplitudes of capsaicin-induced currents before and after nicotine application (mean amplitude of the current preceding nicotine application, 480 ± 70 pA). Note again absence of current recovery. E, effects of different treatments on τ of capsaicin-evoked currents. Same protocol as A. Values were obtained averaging the normalized τ from the first five currents following the treatment. *P < 0.05.

Figure 5. Effect of the chronic application of nicotine on the capsaicin-induced Ca²⁺ transients

A, typical Ca²⁺ transients induced by a 1 s pulse of 1 μm capsaicin in a control DRG neurone (black line) and in a DRG neurone incubated for 18 h with 200 nm nicotine (grey line). B, histogram of the mean amplitude of capsaicin-induced Ca²⁺ transients upon indicated treatments. Δ[Ca²⁺]_i=[Ca²⁺]_i,peak−[Ca²⁺]_i,basal. C, histogram of rise time of capsaicin-induced Ca²⁺ transients upon indicated treatments. *P < 0.05.

Figure 6. Comparison of nAChR P_f values determined in heterologous systems and DRG neurones

Histogram of P_f values of heterologously expressed nAChRs (filled columns) and nAChRs expressed in native DRG neurones (open columns), as indicated. h, human; r, rat. For the P_f values of h α3β4- and h α4β2-nAChRs (expressed in HEK293 cells) see Lax et al. (2002). For the P_f values of h and r α7-nAChRs (expressed in GH4C1 cells) see Fucile et al. (2003). ‘Mixed’ indicates P_f values obtained by activating all nAChR subtypes present in the examined adult DRG neurones. The P_f of α7*-nAChRs was measured in neonatal DRG neurones.