The unanticipated complexity of the selectivity-filter glutamates of nicotinic receptors

Abstract

In ion channels, 'rings' of ionized side chains that decorate the walls of the permeation pathway often lower the energetic barrier to ion conduction. Using single-channel electrophysiological recordings, we studied the poorly understood ring of four glutamates (and one glutamine) that dominates this catalytic effect in the muscle nicotinic acetylcholine receptor ('the intermediate ring of charge'). We show that all four wild-type glutamate side chains are deprotonated in the range of 6.0-9.0 pH, that only two of them contribute to the size of the single-channel current, that these side chains must be able to adopt alternate conformations that either allow or prevent their negative charges from increasing the rate of cation conduction and that the location of these glutamate side chains squarely at one of the ends of the transmembrane pore is critical for their largely unshifted pK(a) values and for the unanticipated impact of their conformational flexibility on cation permeation.

Figure 1. The intermediate ring of glutamates

a, Single-channel current–voltage (i–V) relationships (cell-attached configuration; 1 μM ACh; pH_pipette 7.4) recorded from AChRs having zero, one, two, four, or five glutamates in the intermediate ring, as indicated in the text. Solution compositions are indicated in Supplementary Table 2 (solutions 1 and 2). To increase the mean duration of bursts of openings, the εT264P mutation (at position 12′ of the M2 segment; Supplementary Figs. 1a and 2a) was also engineered. For the “one-glutamate” curve ( , , and ), i–V data from all possible combinations of one glutamate and four alanines around the ring were recorded, and these are indicated with different symbols (one glutamate in either α1 subunit: ; one in β1: ; one in δ: ; one in ε: ). For the data in this graph, whenever the ε subunit carried a (mutant) glutamate at position –1′, the “extra” glycine occupying position –2′ (or –3′; see Supplementary Fig. 1a) was deleted. b, Single-channel inward currents (outside-out configuration; 1μM ACh; solutions 3 and 4) recorded from the adult muscle AChR carrying the burst-prolongingεT264P mutation. The pH values of the extra- and intracellular solutions were the same. For the traces shown, the applied potential was −110 mV at pH 6.0, −100 mV at pH 7.4 and −80 mV at pH 9.0. Openings are downward deflections. Display f_c ≅ 6 kHz.

Figure 2. Mutations reveal a complex conductance behavior

a, b, Single-channel inward currents (cell-attached configuration; ~−100 mV; 1 μM ACh; pH_pipette 7.4; solutions 1 and 2) recorded from AChR mutants containing different residues at position –1′; these are indicated using the single-letter code in the order: α1, ε, α1, β1 and δ subunits (see Supplementary Fig. 1b). All constructs also carried the εT264P mutation. We refer to the current level having a wild-type-like conductance as the “main level”. Openings are downward deflections. Display f_c ≅ 6 kHz. c, Single-channel i–V curves recorded from the indicated six constructs under the same conditions as in a and b. The slopes of the main-level i–V curves are very similar to that of the two-glutamate AAAEE construct (~140 pS; superimposed black dashed line; see Fig. 1a), whereas the slopes of the sublevel i–V curves are very similar to that of AChRs bearing a single glutamate (the rest being alanines) in the intermediate ring (~80 pS; superimposed black dashed line; see Fig. 1a).

Figure 3. The pH dependence of intermediate-ring mutants is weak

a, Single-channel inward currents (outside-out configuration; −100 mV; 1 μM ACh; symmetrical pH; solutions 3 and 4). The construct also carried the εT264P mutation. b, Single-channel inward currents (cell-attached configuration; ~−100 mV; 1 μM ACh; solutions 1 and 2) recorded from the indicated mutant at position 13′ of the δ subunit. The indicated pH values are those of the pipette solution; for this mutant, bathing both sides of the membrane with solutions of the same pH was not necessary to reveal strong pH dependence. To increase the signal-to-noise ratio, the construct also carried two mutations in the ε subunit: a glutamine-to-glutamate mutation at position –1′ and the deletion of the “extra” glycine at position –2′. These ε-subunit mutations increase the single-channel conductance by ~50 pS (“superlevel 1”). The deprotonation of the glutamate engineered at δ13′ increases the conductance even further, by another step of ~50 pS (“superlevel 2”). For both, panels a and b, openings are downward deflections, and display f_c ≅ 6 kHz. c, pH dependence of open-channel current fluctuations recorded from a mutant AChR containing the indicated residues at positions –5′ and –1′ in the background of the εT264P mutant ( ; outside-out configuration; −150 mV; 1 μM ACh; symmetrical pH; solutions 3 and 5) and from the channel with a glutamate engineered at position δ13′ ( ; cell-attached configuration; ~−100 mV; 1 μM ACh; solutions 1 and 2). Data corresponding to open-channel current fluctuations recorded from two δM2 lysine mutants ( and ; outside-out configuration; −100 mV; 1 μM ACh; symmetrical pH; solutions 3 and 5) are also included, for comparison.

Figure 4. The conformational dynamics are sensitive to the local electrostatics

a, The effect of varying the number of aspartates at positions –5′ and –4′ of the M1–M2 linkers on the relative occupancies of the high and low open-channel conductance levels was estimated at ~−100 mV on AChRs having a mutant QQQEE intermediate ring (cell-attached configuration; 1 μM ACh; pH_pipette 7.4; solutions 1 and 2). As is the case for position –1′, the residues occupying positions –5′ and –4′ are indicated using the single-letter code in the order: α1, ε, α1, β1 and δ subunits. Thus, the wild-type sequence is DQDDD at position –5′ and SASAC at position –4′. b, The effect of the membrane potential on the relative occupancies of the high and low open-channel conductance levels was estimated on the AChR having wild-type residues at positions –5′ and –4′, and a mutant QQQEE intermediate ring (cell-attached configuration; 1 μM ACh; pH_pipette 7.4; solutions 1 and 2). The membrane potential (“voltage”) is indicated as the potential on the intracellular side of the patch of membrane minus that on its extracellular side. The fit of the datapoints with a mono-exponential function [Ratio = Ratio_V=0exp(δFV/RT), where V is the membrane potential, and F, R and T are Faraday’s constant, the gas constant, and the absolute temperature, respectively] suggests that, at zero voltage, the plotted ratio of occupancy probabilities is ~6.3 and that the negative charge of the glutamate side chain traverses ~15% (δ = 0.15) of the electric field upon switching between the “up” and “down” conformations. All constructs also carried the εT264P mutation.

Figure 5. Moving the intermediate ring of glutamates into the pore

a, b, Two rotamers of glutamate. Using a model of the bacterial nicotinic-receptor-like GLIC channel (pdb code: 3EAM; ref. 28), a glutamate was “engineered” at position –1′ (a) or 2′ (b) using Coot and VMD molecular-graphics software (in GLIC, the wild-type glutamates of the intermediate ring occur at position –2′). The two conformers were chosen arbitrarily from the library of Lovell and coworkers (see also Supplementary Fig. 5) and are merely meant to illustrate two extreme positions that the Oε1/Oε2 atoms could adopt. In going from position –1′ (a) to 2′ (b), it seems as though the glutamate would lose the ability to alternately “expose” and “hide” the negative charge to and from the pore; the charge would always be inside the pore. c, d, e, Single-channel inward currents (cell-attached configuration; ~−100 mV; 1 μM ACh; solutions 1 and 2) recorded from mutants containing the indicated residues at positions –1′ and 2′ (the latter, also in the order: α1, ε, α1, β1 and δ subunits). The indicated pH values are those of the pipette solution. The three constructs also carried theεT264P mutation. The current-amplitude scale is the same for all three panels. Openings are downward deflections. Display f_c ≅ 6 kHz. We could not record currents from the mutant containing a full ring of alanines at position –1′ and a full ring of glutamates at position 2′ (~100 successful gigaohm seals with no channel activity).