Neuronal nicotinic threonine-for-leucine 247 alpha7 mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Abstract

Mutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric alpha7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We show here that acetylcholine (AcCho) activates two classes of single channels with conductances of 44 pS and 58 pS, similar to those activated by 5HT. However, the mean open time of AcCho-gated ion channels (11 ms) is briefer than that of 5HT-gated ion channels (18 ms). Furthermore, whereas the open time of AcCho channels lengthens with hyperpolarization, that of 5HT channels is decreased. In voltage-clamped oocytes, the apparent affinity of the alpha7 mutant receptor for 5HT is not modified by the presence of dihydro-beta-erythroidine, which acts on the AcCho binding site in a competitive manner. This indicates a noncompetitive action of 5HT on nicotinic acetylcholine receptors. Considered together, our findings show that AcCho gates alpha7 mutant channels with similar conductance but with different kinetic profile than the channels gated by 5HT, suggesting that the two agonists act on different docking sites. These results will help to understand the crosstalk between cholinergic and serotonergic systems in the central nervous system.

Figure 1

Properties of L247T α₇ nAcChoR-channels activated by the natural transmitter AcCho. All panels refer to cell-attached patches of oocytes injected with chicken L247T α₇ subunit cDNA. (A) Examples of single-channel currents at −70 mV EMP. Inward currents are represented by upward deflections. (B) Distribution of single-channel amplitudes at − 53 mV EMP, with 200 nM ACh in the patch pipette, in the same patch. Histogram is best fitted by a single Gaussian curve, with a mean of 2.42 ± 0.02 pA (n = 379). (C) Mean channel current amplitudes at different potentials plotted vs. pipette potential, and fitted by linear regression (solid line), yielding the slope conductance indicated. (D) Histogram of open durations fitted by the sum of two exponential functions, with τ₁ = 2.69 ± 0.01 (67%), τ₂ = 34.84 ± 0.01 (32%), and τ_op as indicated. Same patch as A–C. (E) Voltage-dependence of mean open time obtained from six patches (six cells, two donors). Averaged values of τ_op plotted vs. membrane potential. Error bars indicate SEM. The solid lines represent the least-squares fits of τ_op with the equation τ_op = τ₀ exp(mV), where m indicates the slope of the linear fit. Note the increase of τ_op with membrane hyperpolarization.

Figure 2

Cumulative histograms of single-channel slope conductances. AcCho- and 5HT-gated channel conductances obtained in the cell-attached mode from 18 and 19 oocytes (10 donors), respectively. The histograms are best fitted by the sum of two Gaussian curves, with indicated mean conductances. Only determinations based on a minimum of five holding potentials were included. Ordinate represents number of patches in which each conductance value was observed.

Figure 3

Membrane currents from voltage-clamped oocytes injected with L247T α₇ subunit cDNA. (A) Records of 5HT currents (I_5HT) in a control (Left) and in an oocyte (Right) exposed to 0.1 μM DHβE before applying 5HT at the indicated doses (in μM). Note the current elicited by DHβE, before the 5HT current. Inward currents are represented by downward deflections. The timings of drug applications are indicated by bars above the current traces. (B) 5HT dose-I_5HT relationship fitted to Eq. 1 (see Materials and Methods), in the presence of 0.1 μM DHβE (•) and in standard solution (○). Oocytes were held at −50 mV and exposed to DHβE for 20 s before eliciting I_5HT in the presence of the drug. The current elicited by DHβE peaked to 90 (± 20) nA. The peak I_5HT was normalized to that evoked by 0.5 mM 5HT (I_5HT peak amplitude in standard solution: −1,490 ± 770 μA; mean ± SD). Each point represents the mean ± SD (n = 6; two donors for •; n = 8, three donors for ○). Control: EC₅₀ = 20 μM; n_H = 1.8. In the presence of DHβE: EC₅₀ = 15 μM; n_H = 1.8. Same values were obtained in oocytes voltage-clamped at −110 mV.

Figure 4

Properties of L247T α₇ nAcChoR-channels activated by 5HT. (A) Examples of single-channel currents at −59 mV EMP. (B) Distribution of single-channel amplitudes at same membrane potential, with 20 μM 5HT in the patch pipette, in the same patch. Histogram best fitted as in Fig. 1B, with mean of 2.72 ± 0.02 pA (n = 573). (C) Mean channel current amplitudes vs. pipette potential, plotted and fitted as in Fig. 1C, yielding the indicated slope conductance. Same patch as A and B. (D) Histogram of open durations fitted as Fig. 1D, with τ₁ = 5.04 ± 0.02 (36%), τ₂ = 27.4 ± 0.01 (64%), and τ_op as indicated. Same patch as A–C. (E) Voltage dependence of mean open time obtained from five patches (five cells, three donors). Values of τ_op, errors, and plot, as in Fig. 1E. Note the decrease of τ_op with hyperpolarization.