Loss of Choline Agonism in the Inner Ear Hair Cell Nicotinic Acetylcholine Receptor Linked to the α10 Subunit

Abstract

The α9α10 nicotinic acetylcholine receptor (nAChR) plays a fundamental role in inner ear physiology. It mediates synaptic transmission between efferent olivocochlear fibers that descend from the brainstem and hair cells of the auditory sensory epithelium. The α9 and α10 subunits have undergone a distinct evolutionary history within the family of nAChRs. Predominantly in mammalian vertebrates, the α9α10 receptor has accumulated changes at the protein level that may ultimately relate to the evolutionary history of the mammalian hearing organ. In the present work, we investigated the responses of α9α10 nAChRs to choline, the metabolite of acetylcholine degradation at the synaptic cleft. Whereas choline is a full agonist of chicken α9α10 receptors it is a partial agonist of the rat receptor. Making use of the expression of α9α10 heterologous receptors, encompassing wild-type, heteromeric, homomeric, mutant, chimeric, and hybrid receptors, and in silico molecular docking, we establish that the mammalian (rat) α10 nAChR subunit underscores the reduced efficacy of choline. Moreover, we show that whereas the complementary face of the α10 subunit does not play an important role in the activation of the receptor by ACh, it is strictly required for choline responses. Thus, we propose that the evolutionary changes acquired in the mammalian α9α10 nAChR resulted in the loss of choline acting as a full agonist at the efferent synapse, without affecting the triggering of ACh responses. This may have accompanied the fine-tuning of hair cell post-synaptic responses to the high-frequency activity of efferent medial olivocochlear fibers that modulate the cochlear amplifier.

Keywords: acetylcholine; choline; cochlea; evolution; hearing; ion channels; nicotinic receptors.

Figure 1

Responses of rat and chicken recombinant heteromeric α9α10 nAChRs to nicotinic agonists. (A) Top panel: representative traces of responses to 10 μM ACh (left) blocked by co-application of 100 μM nicotine (right) for rat (black traces) and chicken (red traces) α9α10 receptors. Bottom panel: inhibition curves for nicotine for rat and chicken α9α10 receptors. Responses to 10 μM ACh, co-applied with increasing concentrations of nicotine were normalized to the responses to 10 μM ACh alone. Values are mean ± SEM for six to seven oocytes. (B–D) Top panels: representative maximal responses to ACh and carbachol (B) DMPP (C) and choline (D) for rat (black) and chicken (red) α9α10 receptors. Bottom panels: concentration-response curves for ACh (dotted lines) and carbachol (B) DMPP (C) and choline (D; solid lines) for rat (black) and chicken (red) α9α10 receptors. Values are normalized to the maximal response to ACh obtained in each oocyte. Values are mean ± SEM for five to nine oocytes.

Figure 2

Responses of hair cells to choline. (A) Representative traces of responses to ACh or choline (Ch) in mouse outer (black, top panel) and chicken short (red, bottom panel) hair cells at a holding potential of –40 mV. (B) Percentage of maximum response evoked by ACh or choline normalized to the maximal peak response to 3 mM ACh for mouse and chicken hair cells. Values are mean ± SEM of six (rat) and four (chicken) experiments per group. (*) Friedman’s test, vs. 3 mM ACh for each species (p < 0.05).

Figure 3

Responses of rat and chicken homomeric α9 or α10 nAChRs to choline. Top panels: representative maximal responses to ACh and choline (Ch) for rat α9 (A) chicken α9 (B) and chicken α10 (C) homomeric receptors. Bottom panels: concentration-response curves for ACh (dotted lines) and choline (solid lines) for rat α9 (A) chicken α9 (B) and chicken α10 (C) homomeric receptors. Values are normalized to the maximal response to ACh obtained in each oocyte. Values are mean ± SEM for four to six oocytes.

Figure 4

Responses of rat-chicken hybrid receptors to choline. (A–B) Top panels: representative maximal responses evoked by saturating concentrations of ACh and choline (Ch) in rat α9–chicken α10 (A) and chicken α9–rat α10 (B) hybrid receptors. Bottom panels: concentration-response curves for ACh (dotted lines) and choline (solid lines). Values are normalized to the maximal response to ACh in each oocyte. Values are mean ± SEM of four to six oocytes per group. (C) EC₅₀ values for choline (top panel) and percentage maximal choline response, normalized to the maximal response elicited by ACh (bottom panel) for rat (R) and chicken (C) heteromeric α9α10 receptors, rat α9, chicken α9, and chicken α10 homomeric receptors, and rat α9–chicken α10 and chicken α9–rat α10 hybrid receptors. (*) Kruskal–Wallis test, vs. rat α9α10 receptor (p < 0.05).

Figure 5

Docking of ACh and choline into the binding sites of rat and chicken α9α10 receptors. (A) Ribbon structure of the extracellular domain of rat α10 and α9 subunits showing an interface formed by the principal (+) component of the α10 subunit and the complementary (−) component of the α9 subunit. The location of the orthosteric binding site with a bound ACh is highlighted by the red box. (B) Detailed view of the binding domain highlighted by the red square in panel (A). ACh (green) and the most frequent orientation of choline (blue) are shown docked into the binding pocket. (C,D) Molecular interactions of ACh (C) and choline (D) with residues in the binding site. Cation-π interactions are illustrated by orange dashed lines and H-bonds by the green dashed lines. (E) Frequency (as a percentage) of the most frequent orientation (top panel) and best binding energy (BBE; bottom panel) obtained for three separate simulations for ACh and choline docking into each of the binding interfaces, comprised of (principal/complementary components) α9/α9, α10/α9, α9/α10, and α10/α10 rat (black) or chicken (red) subunits. Values are mean ± SEM of at least three different docking simulations for each interface.

Figure 6

Responses of rat α10_x chimeric receptor to choline. (A) Diagram of an α10_x chimeric subunit showing the regions corresponding to the α9 subunit sequence (red) and α10 subunit sequence (black). (B) Representative maximal responses to ACh (left) and choline (right) for the α9α10_x chimeric receptor. (C) Concentration response-curves for ACh (dotted lines) and choline (solid lines) for α9α10_x chimeric receptors (red circles) and wild-type α9α10 receptors (black squares). Values were normalized to the maximal response to ACh in each oocyte. Values are mean ± SEM of five to nine oocytes per group.

Figure 7

Responses of the rat α9α10W55T receptor to choline. (A) Diagram of an α10 subunit illustrating the position of the W55T mutation, localized to the complementary component of the agonist binding site within the pentameric assembly (top panel). Representative maximal responses to ACh (left) and choline (right) for the α9α10W55T receptor (bottom panel). (B) Concentration response-curves to ACh (dotted lines) and choline (solid lines) for the α9α10W55T receptor (red circles) and wild-type α9α10 receptors (black squares). Values were normalized to the maximal response to ACh in each oocyte. Values are mean ± SEM of five to nine oocytes per group. (C) Representative responses to 10 μM ACh (dotted lines) alone or on top of responses to 300 or 3,000 μM choline in oocytes expressing wild-type α9α10 or α9α10W55T receptors. (D) Normalized responses to 10 μM ACh obtained in the presence of increasing concentrations of choline (Ch) for wild-type α9α10 and α9α10W55T receptors. Responses were normalized to responses to 10 μM ACh in the absence of choline for each oocyte. (*) Friedman’s test, vs. 10 μM ACh on top of 10 μM Ch (p < 0.05).