Key roles of hydrophobic rings of TM2 in gating of the alpha9alpha10 nicotinic cholinergic receptor

Abstract

We have performed a systematic mutagenesis of three hydrophobic rings (17', 13' and 9') within transmembrane region (TM) 2 of the alpha9alpha10 nicotinic cholinergic receptor (nAChR) to a hydrophilic (threonine) residue and compared the properties of mutant receptors reconstituted in Xenopus laevis oocytes. Phenotypic changes in alpha9alpha10 mutant receptors were evidenced by a decrease in the desensitization rate, an increase in both the EC(50) for ACh as well as the efficacy of partial agonists and the reduction of the allosteric modulation by extracellular Ca(2+). Mutated receptors exhibited spontaneous openings and, at the single-channel level, an increased apparent mean open time with no major changes in channel conductance, thus suggesting an increase in gating of the channel as the underlying mechanism. Overall, the degrees of the phenotypes of mutant receptors were more overt in the case of the centrally located V13'T mutant. Based on the atomic model of the pore of the electric organ of the Torpedo ray, we can propose that the interactions of side chains at positions 13' and 9' are key ones in creating an energetic barrier to ion permeation. In spite of the fact that the roles of the TM2 residues are mostly conserved in the distant alpha9alpha10 member of the nAChR family, their mechanistic contributions to channel gating show significant differences when compared to other nAChRs. These differences might be originated from slight differential intramolecular rearrangements during gating for the different receptors and might lead each nAChR to be in tune with their physiological roles.

Figure 1

Responses of mutant receptors to ACh. (a) Alignment of the amino-acid sequences of the Torpedo α, rat α1, α7, α9 and α10 nAChR subunits. Residues that have been mutated are shown in bold. (b) Representative responses to increasing concentrations of ACh for wild-type and each mutant receptor. (c) Concentration–response curves to ACh. Peak current values were normalized and referred to the maximal peak response to ACh in each case. The mean and s.e.m. of four to five experiments per group are shown. (d) Representative responses of wild-type and mutant receptors to a 1-min application of 100 μM ACh.

Figure 2

Modulation of ACh responses by extracellular calcium. (a) Bar diagram illustrating the effects of extracellular Ca²⁺ on responses to ACh in wild-type and mutant receptors at a membrane holding potential of −90 mV. The concentration of ACh used in each case was near the corresponding EC₅₀ value derived from the concentration–response curves of Figure 1: 10 μM for the wt, 0.5 μM for the 9′ and 0.1 μM for the 13′ mutant. Current amplitudes obtained at different Ca²⁺ concentrations in each oocyte were normalized with respect to that obtained at 1.8 mM in the same oocyte. Each bar represents the mean and s.e.m. of the normalized response obtained in different oocytes (n=4–10 per bar). ^*P<0.05 with respect to the corresponding value at 0.1 mM Ca²⁺. (b) Representative I–V curves, obtained by application of a voltage ramp protocol (−120 to +50 mV, 2 s) 10 s after the peak response to either 0.5 μM ACh for the 9′ (upper panel, n=6) or 0.1 μM ACh for the 13′ mutant (lower panel, n=6). Oocytes were voltage-clamped at −70 mV, and ramps were performed at different Ca²⁺ concentrations. (c) Concentration–response curves to ACh, performed either at nominally zero or 1.8 mM Ca²⁺. Responses were normalized to the maximum obtained at 1.8 mM Ca²⁺ for each case. The mean and s.e.m. of four to 10 experiments per group are shown. ^*P<0.05 with respect to the corresponding value at nominally zero Ca²⁺.

Figure 3

Choline is a full agonist of mutant receptors. Concentration–response curves to choline were performed. Peak current values were normalized and referred to the maximal peak response to ACh in each case. The mean and s.e.m. of four and seven experiments for wild-type and V13′T receptors, respectively, are shown. The EC₅₀ and Hill coefficients are shown in Table 2.

Figure 4

Effect of classical antagonists of the α9α10 nAChR. Concentration–response curves to ACh, ICS 205,930, muscarine and nicotine were performed. Peak current values were normalized and referred to the maximal peak response to ACh in each case. The mean and s.e.m. are shown. The number of experiments for each set of data is shown in Table 2. The EC₅₀, Hill coefficients and maximal responses are shown in Table 2.

Figure 5

Block of leak current by strychnine. (a) Representative responses (n=5 per mutant) to strychnine of oocytes injected with either the 9′ (upper panel) or the 13′ (lower panel) mutant receptors. Note the deflection of currents in the upward direction in the presence of the drug. A maximal response to ACh in each oocyte is shown for comparison. (b) Correlation of the EC₅₀ values for ACh for each receptor with the degree of spontaneous activity calculated as the percentage of the maximal response to strychnine compared to that of ACh (r²: 0.989).

Figure 6

Single-channel recordings of wild-type and mutant α9α10 nAChRs. (a) (left) Channel traces recorded in the cell-attached configuration from oocytes injected with wild-type, L9'T and V13'T subunits. As a control, endogenous channels were recorded from noninjected oocytes. Traces are shown at two different time scales for each recording. Currents are displayed at a bandwidth of 5 kHz with channel openings as upward deflections. Pipette potential: 120 mV. To the right, open-time and amplitude histograms of the corresponding recordings are shown. (b) Channel traces obtained after application of ACh to outside-out patches from oocytes injected with wild-type or V13′T α9α10 subunits. Currents are displayed at a bandwidth of 5 kHz with channel openings as downward deflections. Pipette potential: −70 mV.