Tunable pKa values and the basis of opposite charge selectivities in nicotinic-type receptors

Abstract

Among ion channels, only the nicotinic-receptor superfamily has evolved to generate both cation- and anion-selective members. Although other, structurally unrelated, neurotransmitter-gated cation channels exist, no other type of neurotransmitter-gated anion channel, and thus no other source of fast synaptic inhibitory signals, has been described so far. In addition to the seemingly straightforward electrostatic effect of the presence (in the cation-selective members) or absence (in the anion-selective ones) of a ring of pore-facing carboxylates, mutational studies have identified other features of the amino-acid sequence near the intracellular end of the pore-lining transmembrane segments (M2) that are also required to achieve the high charge selectivity shown by native channels. However, the mechanism underlying this more subtle effect has remained elusive and a subject of speculation. Here we show, using single-channel electrophysiological recordings to estimate the protonation state of native ionizable side chains, that anion-selective-type sequences favour whereas cation-selective-type sequences prevent the protonation of the conserved, buried basic residues at the intracellular entrance of the pore (the M2 0' position). We conclude that the previously unrecognized tunable charge state of the 0' ring of buried basic side chains is an essential feature of these channels' versatile charge-selectivity filter.

Figure 1. The versatile charge selectivity of nicotinic-type receptors

a, Sequence alignment of residues in and flanking the M1–M2 loop. The broken horizontal line separates the sequences that are known or predicted (on the basis of their sequences) to form cation-selective channels (top) from those that are known or predicted to form anion-selective ones (bottom). Included in this alignment are subunits from receptors to acetylcholine (ACh), serotonin (5-HT), glycine (Gly), γ-aminobutyric acid (GABA), glutamate (Glu), histamine (His), and from receptors with as yet unidentified ligands. The invertebrate organisms in this list are: Aplysia californica (a mollusc), the fruit fly Drosophila melanogaster (an arthropod), Caenorhabditis elegans (a nematode), Lymnaea stagnalis (a mollusc), Capitella capitata (an annelid), Schistosoma mansoni and S. haematobium (two human parasitic platyhelminths), and Helobdella robusta (an annelid). b, Macroscopic current–voltage (I–V) relationships recorded under KCl-dilution conditions (solutions 8 and 9 in Supplementary Table 1; pH 7.4, both sides) in the outside-out configuration, as indicated in Supplementary Fig. 9 and in Methods. The equilibrium (Nernst) potentials at 22°C, using ion concentration values, are −55.0 mV for K⁺ and +50.6 mV for Cl⁻. Reversal potentials are indicated.

Figure 2. A proline mutation unveils a proton-binding site

a, Single-channel inward currents (cell-attached configuration; ~ −100 mV; 1 μM ACh; pH_pipette 7.4) recorded from a mutant AChR having a proline inserted between positions −2′ and −1′ of the δ subunit. To increase the number of main-level ⇄ sublevel interconversions, a mutation that prolongs the mean duration of bursts of openings (εT264P) was also engineered. Solution compositions are indicated in Supplementary Table 1 (solutions 1–3). Mutations are indicated on the M1–M2 loop sequences; underlined bold symbols denote insertions whereas bold symbols (without the underline) denote substitutions. b, Inward currents (cell-attached configuration; ~ −100 mV; 1 μM ACh; pH_pipette 7.4; solutions 2 and 3) recordedfrom the indicated AChR constructs. The burst-prolonging mutation was εT264P (in the case of AChRs with a proline inserted in the α1, β1 or δ subunit) or δS268Q (in the case of the glycine-to-proline substitution mutant at position −2′ of the ε subunit). In the case of the α1-subunit insertion, the trace shown corresponds to the construct having only one of the two α subunits mutated. c, Single-channel I–V relationships (cell-attached configuration; 1 μM ACh; pH_pipette 7.4; solutions 2 and 3) recorded from the five constructs in b. For clarity, only the I–V curves corresponding to the sublevel are shown for the mutants. To facilitate the visual comparison of the slopes, each curve was displaced along the voltage axis so that it extrapolates exactly to the origin. d, pH dependence of the main-level ⇄ sublevel current fluctuations (outside-out configuration; −100 mV; 1 μM ACh; solutions 4 and 5).

Figure 3. The side chain of the 0′ basic residue is the proton-binding site

a, Single-channel inward currents (cell-attached configuration; 1 μM ACh; pH_pipette 7.4; solutions 2 and 3) recorded from AChRs with a proline inserted between positions −2′ and −1′ of the δ subunit and having four of the five native ionizable residues that flank δM2 mutated to alanine, one at a time. The burst-prolonging mutation was εT264P. Mutation of the fifth residue (the 0′ lysine) to alanine, glutamine or valine (in the presence of the inserted proline) abolishes receptor expression on the plasma membrane, as revealed by the lack of specific α-bungarotoxin binding. The applied potential was ~ −100 mV for all constructs, with the exception of the receptor containing the glutamate-to-alanine mutation at position −1′, in which case the potential was ~ −150 mV (to compensate for its lower single-channel conductance). b, Inward currents recorded from a mutant AChR having a proline inserted between positions −2′ and −1′ of the β1 subunit and from the mutant having, in addition, a lysine-to-glutamine mutation at position 0′ of the same subunit. The applied potential was ~ −100 mV. All other experimental conditions were as in a.

Figure 4. Not only prolines, not only insertions

a, Single-channel inward currents (cell-attached configuration; ~ −100 mV; 1 μM ACh; pH_pipette 7.4; solutions 2 and 3) recorded from the indicated AChR insertion mutants. The burst-prolonging mutation was εT264P. Threonine insertions have a similar effect. b, Inward currents recorded from the indicated AChR substitution mutants under the same experimental conditions as in a. Note that the insertion of a residue is not required to reveal a proton-binding site. Instead, replacing the conserved glycine at position −2′ with a variety of other residues (see Supplementary text; only proline is shown, here) also unveils a protonation site in the four types of subunit. In the case of the α1-subunit mutant, the trace shown corresponds to the construct having only one of the two α subunits mutated. The trace illustrating the effect of a glycine-to-proline mutation at this position of the ε subunit is shown in Fig. 2b.