Stoichiometry for activation of neuronal α7 nicotinic receptors

Abstract

Neuronal α7 nicotinic receptors elicit rapid cation influx in response to acetylcholine (ACh) or its hydrolysis product choline. They contribute to cognition, synaptic plasticity, and neuroprotection and have been implicated in neurodegenerative and neuropsychiatric disorders. α7, however, often localizes distal to sites of nerve-released ACh and binds ACh with low affinity, and thus elicits its biological response with low agonist occupancy. To assess the function of α7 when ACh occupies fewer than five of its identical binding sites, we measured the open-channel lifetime of individual receptors in which four of the five ACh binding sites were disabled. To improve the time resolution of the inherently brief α7 channel openings, background mutations or a potentiator was used to increase open duration. We find that, in receptors with only one intact binding site, the open-channel lifetime is indistinguishable from receptors with five intact binding sites, counter to expectations from prototypical neurotransmitter-gated ion channels where the open-channel lifetime increases with the number of binding sites occupied by agonist. Replacing the membrane-embedded domain of α7 by that of the related 5-HT3A receptor increases the number of sites that need to be occupied to achieve the maximal open-channel lifetime, thus revealing a unique interdependence between the detector and actuator domains of these receptors. The distinctive ability of a single occupancy to elicit a full biological response adapts α7 to volume transmission, a prevalent mechanism of ACh-mediated signaling in the nervous system and nonneuronal cells.

Keywords: Cys-loop receptors; agonist binding site; channel gating; patch-clamp; α7 nicotinic receptor.

Fig. 1.

Macroscopic responses of α7 with different average number of functional binding sites per receptor. Representative macroscopic responses to the application of a 300-ms pulse of 100 μM (gray line) or 1 mM ACh (black line) of cells expressing α7 (A) or receptors formed by α7 and α7-Y188T (subunit ratio 1:3) (C). No responses are elicited by 1 mM ACh from cells expressing α7-Y188T receptors (B). (D) Dose–response curves for α7 (●) and α7 + α7-Y188T receptors [1:3 () and 1:9 subunit ratios (○)]. Each point corresponds to the mean ± SD.

Fig. 2.

Single-channel currents from cells transfected with α7 or mixed α7 + α7-Y188T subunits. Single-channel currents from cells transfected with α7 (A) or α7 and α7-Y188T (subunit ratio 1:3) (B) were recorded at −70 mV (500 μM ACh). Channel traces are shown at two different time scales. Typical open and burst duration histograms are shown for each condition. Openings are shown as upwards deflections.

Fig. 3.

Electrical fingerprinting for α7. (A) Single-channel currents activated by 100 μM ACh from wild-type α7. Amplitude histogram constructed with events longer than 0.3 ms. (B) Single-channel currents activated by 100 μM ACh from cells transfected with α7TSLMF alone (Upper) or together with α7TSLMF_LC (Lower) (subunit ratio 1:1). (C) Single-channel currents activated by 100 μM ACh in the presence of 2 mM 5-HI from cells transfected with α7 alone (Upper) or together with α7_LC (Lower) (subunit ratio 1:1). The two traces for the mixed subunits are excerpts from the same recording. Representative amplitude histograms are shown. Membrane potential: −70 mV. (D) Homology model of a single α7 subunit based on the Torpedo AChR structure (PDB ID code 2BG9), with the mutations that lead to the low-conductance form of α7 (α7_LC). (E) Plot of mean current amplitude against number of mutant low-conductance subunits. The fitted slopes are: 1.89 pA/LC subunit for α7 + α7_LC in the presence of ACh + 5-HI and 1.97 pA/LC subunit for α7TSLMF + α7TSLMF_LC in the presence of ACh. Note that α7 wild-type conductance is referred to as high conductance.

Fig. 4.

Comparison of mean open and burst durations for α7 receptors containing five or one functional binding sites. Cells were transfected with α7TSLMF_LC and α7TSLMF carrying (Lower) or not (Upper) the Y188T mutation (A) or with α7_LC together with α7 (Upper) or α7-Y188T (Lower) (B). Single channels activated by 100 μM ACh in the absence (A) or presence of 2 mM 5-HI (B). The 8-pA amplitude class corresponds to receptors containing five or only one functional binding site (in the presence of Y188T mutation). Open and burst duration histograms are shown for each condition. Membrane potential: −70 mV.

Fig. 5.

Duration of bursts of α7–5-HT₃A containing five or one functional binding sites. Cells were cotransfected with control (α7–5HT₃A) and high-conductance (α7–5HT₃A_HC) subunits carrying (B) or not (A) Y188T mutation. Single channels were recorded in the presence of 1 mM ACh and 2 mM 5-HI at −120 mV. Channels of the amplitude class determined by four high-conductance subunits were analyzed, which correspond to arrangements containing five (A) or one (B) functional binding sites.