SUPPLEMENTARY INFORMATION

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1 doi: /nature10015 SUPPLEMENTARY TEXT The nicotinic-receptor superfamily of ion channels and their versatile charge-selectivity filter Three structurally unrelated groups of non-selective cation channels support ionotropic excitatory signals, namely, the superfamilies of nicotinic-type receptors, excitatory glutamate receptors and ATP P2X receptors. Of these, only the nicotinic-receptor superfamily has evolved to also generate highly anion-selective channels, which (depending on the ratio of Cl concentrations across the membrane, among other factors) can mediate synaptic inhibition or excitation In vertebrates, receptors to acetylcholine (ACh) or serotonin (5-HT) form cation-selective channels whereas receptors to γ-aminobutyric acid (GABA) or glycine (Gly) form anion-selective ones. In invertebrates, receptors to a much wider variety of ligands (including not only ACh, 5-HT and GABA, but also, glutamate 15, histamine 36, tyramine 37,38 and dopamine 38 ) and receptors displaying ligand-specificity/charge-selectivity combinations not seen among vertebrates (such as anion-selective ACh 16,39 and 5-HT receptors 40, as well as cation-selective GABA receptors 17,41 ) have been described. Mutational studies of the different members of the superfamily have revealed that native ionizable residues in regions other than the intracellular end of M2 can also affect the single-channel conductance However, only the mutations at or near positions 1 and 2 discussed here have been shown to change the charge selectivity of these channels, as judged from reversal-potential measurements 1 10, As an example, see Fig. 1b for the effect of deleting the five prolines at position 2 of the α1 GlyR on anion selectivity (see also ref. 6). The side chains of the buried lysines at position 0 are deprotonated in the wild-type AChR We previously proposed that the εnh 2 group of the δ- subunit s 0 lysine is deprotonated 12. We based this conclusion on the observation that a lysine-to-alanine mutation at this position of the δ subunit does not result in an increased single-channel conductance (as could be expected if this lysine s side chain were positively charged) and on the current-blocking effect of lysines, arginines and histidines substituted at approximately ninety other positions in and flanking M1, M2 and M3 (refs 12, 13). Furthermore, we reported that the microenvironment around this position seems to be so hydrophobic (and, hence, so inhospitable for charges, regardless of their sign) that even the highly basic guanidine group of an engineered arginine (pk a bulk, arginine 12.5; pk a bulk, lysine 10.4) fails to reduce the single-channel conductance at ph 6.0 (ref. 12). It could be argued, though, that the AChR s singlechannel conductance may be close to some sort of upper limit, and hence, that the removal of a positively-charged side chain from the M2 segment cannot be expected to increase the amplitude of unitary currents to a detectable extent. However, the observation that the negatively-charged side chain of a deprotonated aspartate or glutamate engineered at pore-lining positions does cause an increase in the single-channel conductance (Supplementary Fig. 4) should dispel this concern. Still, it could be speculated that, being in the backside of M2, the side chain of the 0 lysine may be too far from the long axis of the pore for its positive charge to affect the rate of ion conduction. However, the current-attenuating effect of lysines engineered to be on the same stripe of the α helix as the native 0 lysine (for example, at position 11 ; Supplementary Fig. 4) suggests that this is not the case. Unusually acidic, deprotonated lysine or arginine side chains are not unprecedented. For example, the pk a of the Schiff-base-forming lysine at the active site of the enzyme acetoacetate decarboxylase was reported to be ~5.9 (ref. 48) whereas that of the proton-abstracting arginine at the active site of family 1 pectin lyase A was suggested to be ~6.0 (ref. 49). And in the AChR s δ subunit, lysines engineered at positions on the backside of M2 (such as 3, 4, 7 and 11 ) also have highly depressed pk a s 12. In this study, we report that the same is the case for the 0 lysines in the β1 and ε subunits in the sense that their mutation to alanine (in the background of the wild-type AChR) does not lead to an increased single-channel conductance, either (Supplementary Fig. 5a and Supplementary Table 2). Thus, these side chains also seem to be facing away from the pore s lumen, buried in their corresponding M1 M2 M3 interfaces and unable to bind protons. In addition, we found that mutation of these lysines to alanine in the β1, δ or ε subunits has little impact on the kinetics of deactivation, entry into desensitization and recovery from desensitization (Supplementary Fig. 5b e and Supplementary Table 2) suggesting only a minor effect on the underlying rate constants. Mutation of the two 0 lysines in the α1 subunits to alanine, cysteine or glutamine, however, reduces the expression of the receptor in the plasma membrane (Supplementary Fig. 5f) to the extent that currents could not be recorded. An anomalous interaction with Ca 2+ and Mg 2+ The relatively simple effect of Ca 2+ and Mg 2+ on ion permeation through the wild-type AChR (Supplementary Fig. 2a), and even through lysine-substituted M2 mutants (Supplementary Figs 2b and 6), contrasts markedly with the more complex interaction of these divalent cations with the M1 M2 loop mutants studied here (Fig. 2a). But, in retrospect, this is not entirely surprising. In fact, it seems reasonable to expect that, however subtle, the rearrangements caused by the insertions and substitutions in the M1 M2 loop result not only in the repositioning of the 0 lysines, but also, in the repositioning of nearby residues. Right next to the 0 lysines, at position 1 (Fig. 1a), is the ring of four glutamates and one glutamine ( the intermediate ring of charge ) that constitutes the primary determinant of divalent-cation permeation through the AChR 50. Ca 2+ and Mg 2+ permeate more slowly than K + or Na + through the wild-type muscle AChR, yet they traverse the channel fast enough for the single-channel current not to drop to zero when the pore is occupied by these divalent cations 51. As a result, the addition of Ca 2+ and Mg 2+ to extracellular solutions containing, primarily, K + or Na + simply attenuates the (inward) single-channel current amplitude (Supplementary Fig. 2a). Furthermore, in the case of mutants with lysines substituted along M2, both the mainlevel and the sublevel conductances are attenuated to comparable degrees (Supplementary Fig. 2b), and thus, 1

2 extent-of-channel-block values in the presence or absence of these extracellular divalent cations do not differ greatly (Supplementary Fig. 6). The interaction of Ca 2+ and Mg 2+ with the mutant AChRs studied here is different in that the sublevel s amplitude is attenuated by these cations to a much lower degree (~16% by 1.8 mm Ca 2+ and ~4.5 % by 1.7 mm Mg 2+ ) than is the main-level s (~37% by Ca 2+ and ~22% by Mg 2+ ) (Fig. 2a). This phenomenon is entirely consistent with the lysine at position 0 lowering the affinity of Ca 2+ and Mg 2+ for the nearby ring of 1 glutamates when the εnh 2 group is protonated. Moreover, we found that millimolar Mg 2+ (but not Ca 2+ ) causes brief and frequent excursions of the singlechannel current to the baseline that, most likely, represent abnormally prolonged sojourns of Mg 2+ in the pore. Kinetic analysis of the current fluctuations associated with the protonation-deprotonation transitions and with these Mg 2+ block-unblock events revealed that this divalent cation is much more likely to block the channel when the 0 lysine is deprotonated. This finding is also fully consistent with the notion of a decreased affinity of divalent cations for the pore when the 0 lysine is protonated. We conclude that, in the same way as the repositioning of the lysines at position 0 increases the affinity of their εnh 2 groups for protons, the concomitant rearrangement of the adjacent carboxylates and the increased local positive potential that results from the protonation of the 0 side chains lead to an altered interaction of divalent cations with the glutamates at position 1. Anion-selectivity without amino-acid insertions Although the amino-acid sequence of the M1 M2 loop is rather variable, the glycine-glutamate-lysine or glycineglutamate-arginine motif near the intracellular end of M2 is very well conserved among the cation-selective channels of vertebrates (Fig. 1a). Invertebrate members of the superfamily, however, display a much wider repertoire of amino acids in this three-residue region to the extent that the only conserved feature seems to be (on the basis of the metazoan genomes sequenced to date) the presence of a lysine or an arginine at position 0. Although the type of charge selectivity conferred by these invertebrate sequences remains largely unexplored, molluscan homomeric AChRs having a proline-alanine-lysine motif (where the proline and the alanine can be regarded as replacing the glycine and the glutamate, respectively, of the glycine-glutamate-lysine motif of vertebrates; Fig. 1a) were found to be highly anion selective (P Cl /PNa + P Cl /PK + 100; ref. 16). Quite notably, we found that glycine-to-proline substitutions engineered at the AChR s 2 position of the different subunits give rise to current fluctuations (Fig. 4b) that resemble those caused by the several insertions characterized here. Furthermore, we found that this effect is not a unique property of proline but that also alanine, serine, asparagine (Supplementary Table 3) and even lysine substitutions (see Supplementary Text, below) have a similar effect when replacing the 2 glycine of the δ subunit. Indeed, it may well be that only in the presence of a glycine at position 2 can the 0 basic side chain remain well-tucked into the hydrophobic interior of the protein, fully deprotonated. Without a doubt, the additional presence of a glutamate-to-alanine substitution in the native proline-alanine-lysine motif of some invertebrate AChRs (Fig. 1a) would accentuate the electrostatic effect of the positive charge on the 0 lysine. In light of these results, we anticipate that other chargeselectivity-filter motifs present in invertebrates would also confer anion-selectivity, even in the absence of an extra residue in the M1 M2 loop. Among these are the alaninealanine-lysine motif of nicotinic-receptor-like sequences from several blood flukes (such as Schistosoma mansoni and S. haematobium) and the asparagine-alanine-lysine motif predicted by the genome of the leech Helobdella robusta (Fig. 1a). Extent of channel block: one more observation In previous work 12,13, we estimated the orientation of transmembrane residues (relative to the long axis of the pore) in the open-channel conformation of the AChR by measuring some properties of systematically engineered basic amino acids. Lysines, arginines or histidines were introduced in the channel by mutating the wild-type sequence, one residue at a time. Because the patterns of extent-of-block and pk a values along the scanned M1, M2 and M3 transmembrane segments were consistent with the α-helical secondary structure revealed by cryo-electron microscopy 52, the mutations were deemed not to distort the channel, at least not to an extent that would invalidate our mutational approach. This notion also led us to assume that the observed protonation-deprotonation events reflect proton-transfer reactions involving only the substituted basic side chains without any contribution from the few native ionizable side chains that flank the mutated α-helices. The finding in this paper that mutations in the AChR s M1 M2 loop can affect the orientation of the side chain of the native lysine at position 0, to the point that its εnh 2 group can become protonated and thus contribute to the extent of block, led us to revisit this assumption. To this end, we engineered lysines along M2 and the M1 M2 loop in the background of an AChR with an alanine at 0, instead. We found that lysine substitutions at positions 2 and 1 of the δ subunit cause more channel block when engineered in the wild-type background than they do in the background of the channel with an alanine at δ0 ; it is these alaninebackground values that are plotted in Supplementary Fig. 6. For positions along M2, however, we found that the extent of block upon lysine substitution is not affected by whether the wild-type lysine or a mutant alanine occupies position

3 a H 3 N + b γ 20 Extracellular COO α1 α1 Transmembrane M1 M2 M3 M4 M1 M2 Intracellular 7 0 M1-M2 loop δ M4 M3 β1 Supplementary Figure 1. Some structural aspects of the nicotinic-receptor superfamily of ion channels. a, Membranethreading pattern common to all the subunits belonging to this superfamily of pentameric ion channels. The approximate location of some amino-acid positions is indicated using the prime-numbering system. The colour code is the same for both panels. b, Ribbon representation of the transmembrane portion of the PDB file 2BG9 (4-Å resolution; ref. 52), a model of Torpedo s electric-organ AChR (a muscle-type AChR) in the absence of activating ligands, as viewed from the extracellular side. The linker between M3 and M4 could not be resolved. In adult muscle, the γ subunit is replaced by the ε subunit. The molecular image was made with VMD (ref. 53). 3

4 a Wild type 1.8 mm Ca 2+, 1.7 mm Mg 2+ no Ca 2+, no Mg 2+ b Shut Sublevel Main level δa10'k 1.8 mm Ca 2+, 1.7 mm Mg 2+ no Ca 2+, no Mg ms 5 pa 25 ms 5 pa Supplementary Figure 2. Interaction between the pore and divalent cations. a, Single-channel inward currents (cellattached configuration; ~ 100 mv; 1 µm ACh; ph pipette 7.4) recorded from the wild-type AChR. Solution compositions are indicated in Supplementary Table 1 (solutions 1 3). Openings are downward deflections. b, Inward currents recorded from a mutant AChR having a lysine substituted at position 10 of the M2 segment of the δ subunit under the same experimental conditions as in a. The burst-prolonging mutation was εt264p. 4

5 a Shut Sublevel αl9'k βl9'k δl9'k εl9'k 15 ms 10 pa b Voltage (mv) Wild type αl9'k βl9'k δl9'k εl9'k Current (pa) Supplementary Figure 3. Nearly symmetric contribution of all five M2 segments to the open-channel pore lining. a, Single-channel inward currents (cell-attached configuration; ~ 190 mv; 1 µm ACh; ph pipette 7.4; solutions 2 and 3) recorded from mutant AChRs having lysines substituted at position 9 of the M2 segment of the different subunits. In the case of the α1-subunit mutation, the trace shown corresponds to the construct having only one of the two α subunits mutated. The openchannel currents seem to dwell exclusively on the sublevel, which indicates that the side chains of lysines engineered at these positions have a high affinity for protons, characteristic of water-exposed εnh 3 + groups. b, Single-channel I V relationships (cell-attached configuration; 1 µm ACh; ph pipette 7.4; solutions 2 and 3) recorded from the four mutants in a and from the wild-type AChR. Only I V curves corresponding to the sublevel could be measured 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. The values of the single-channel conductances are: ± 0.9 ps for the wild type; 25.4 ± 0.7 ps for the α1-subunit mutant; 36.5 ± 0.8 ps for the β1-subunit mutant; 32.9 ± 0.6 for the δ-subunit mutant; and 28.3 ± 0.6 ps for the ε-subunit mutant. Hence, taking the conductance of the wild-type channel as that of the main level of these 9 -position mutants (which cannot be measured accurately even at ph 10.5), the corresponding extent-of-block values are: 0.808, 0.725, and for the α1-, β1-, δ- and ε-subunit mutants, respectively. Clearly, not only the proton affinities, but also, the extent-of-block values corresponding to the 9 lysines in the different subunits are very similar. 5

6 Wild type δs6'e Shut Main level Superlevel Shut δv13'd δq11'k Sublevel Main level 20 ms 10 pa Supplementary Figure 4. Effect of engineered pore-facing acidic and back-facing basic residues in M2. Single-channel inward currents (cell-attached configuration; ~ 100 mv; 1 µm ACh; solutions 2 and 3) recorded from the indicated AChR constructs. The ph of the pipette solution was 7.4 in the case of the wild-type AChR and the two acidic-residue substitution mutants. In the case of the lysine mutant at position 11, however, this ph needed to be lowered to 6.0 to reveal clear sojourns in the sublevel. The highly depressed pk a of this lysine is consistent with the notion that the backside of M2 faces a hydrophobic microenvironment. This hydrophobicity seems to be even more pronounced at the level of the 0 position. Also in the case of the 11 construct, the burst-prolonging mutation was εt264p. Deprotonation of individual acidic residues increases the single-channel conductance by ~40 ps, giving rise to a superlevel. Hence, the wild-type conductance of ~130 ps (under our experimental conditions) does not represent an upper limit (see Supplementary Text). Note that, at ph pipette 7.4, aspartates or glutamates engineered at pore-facing positions of M2 (such as 6 and 13 ) are mostly protonated, and hence, largely neutral. 6

7 a Voltage (mv) b 5 Wild type βk0'a δk0'a εk0'a Current (pa) 2 ms c d Recovered fraction ms 0.0 e f Interval duration (ms) Normalized peak current Pulse number wild type βk0'a δk0'a εk0'a AChR expression in the plasma membrane Wild-type AChR αk0'a αk0'c αk0'q βk0'a δk0'a εk0'a Supplementary Figure 5. Effect of mutations at position 0. a, Single-channel I V relationships (cell-attached configuration; 1 µm ACh; ph pipette 7.4; solutions 2 and 3) recorded from the indicated AChR constructs. To facilitate the visual comparison of the slopes, each curve was displaced along the voltage axis so that it extrapolates exactly to the origin. The values of the single-channel conductances are given in Supplementary Table 2. Currents could not be recorded from AChRs having the 0 lysines in both α1 subunits mutated to non-ionizable amino acids, an effect we ascribe to these mutants 7

8 low expression levels. The colour code is the same for all panels. b, Kinetics of deactivation (outside-out configuration; 80 mv; ph 7.4, both sides; solutions 6 and 7). Each plotted trace is the average response of an outside-out patch to several 0.8- ms pulses of 100 µm ACh applied as low-frequency trains ( Hz). The deactivation time constants, estimated from mono-exponential fits to the decaying phase of these plots, were in turn averaged over several such trains. These values are given in Supplementary Table 2. The traces represent normalized currents. c, Kinetics of entry into desensitization (outsideout configuration; 80 mv; ph 7.4, both sides; solutions 6 and 7). Each plotted trace is the average response of an outside-out patch to several 1- or 2-s pulses of 100 µm ACh applied as low-frequency trains (<0.1 Hz). The entry-into-desensitization time constants, estimated from mono-exponential fits to the decaying phase of these plots, were in turn averaged over several such trains. These values are given in Supplementary Table 2. The traces represent normalized currents. d, Kinetics of recovery from desensitization (outside-out configuration; 80 mv; ph 7.4, both sides; solutions 6 and 7) estimated using pairs of fully-desensitizing (1-s) and test (100-ms) pulses of 100 µm ACh. Error bars are standard errors calculated from the results of several independent experiments (one experiment per patch). The recovery-from-desensitization time constants, estimated from mono-exponential fits to the plotted functions, are given in Supplementary Table 2. e, Response to repetitive stimulation (outside-out configuration; 80 mv; ph 7.4, both sides; solutions 6 and 7). The peak values of currents elicited by 25-Hz trains of 0.8-ms pulses of 100 µm ACh were normalized with respect to the first peak in each series (black circle) and averaged across replicate experiments. Error bars are standard errors. The number of averaged responses is 4 for the wildtype channel; 10 for the β1-subunit mutant; 13 for the δ-subunit mutant; and 5 for the ε-subunit mutant. f, Expression of mutant AChRs in the plasma membrane of transfected HEK-293 cells (relative to the wild-type channel) estimated using [ 125 I]-α-bungarotoxin, as indicated in Methods. Error bars are standard errors. It is clear from this figure that the main effect of replacing the lysine at position 0 with other residues is to reduce the expression of the channel at the cell surface; the receptor s functional properties are affected to a much lesser degree. 8

9 Residue number 24' 22' 20' 18' 16' 14' 12' 10' 8' 6' 4' 2' 0' 2' 5' 7' Transmembrane Extracellular Intracellular Extent of channel block Supplementary Figure 6. Extent-of-block values. Black circles correspond to the blocking effect of single lysines engineered in and around the M2 segment of the δ subunit estimated from cell-attached recordings obtained in the nominal absence of extracellular Ca 2+ or Mg 2+ (solutions 2 and 3); gray circles are their counterparts in M2 in the presence of extracellular 1.8 mm Ca 2+ and 1.7 mm Mg 2+ (solutions 1 and 3; data taken from ref. 12). We attribute the lack of block at positions 0 and 4 (even at ph 6.0) to the highly acidic pk a of basic side chains introduced at these positions rather than to the lack of current-attenuating effect of positive charges engineered in the backside of M2. The red circle corresponds to the block exerted by the side chain of the native 0 lysine upon insertion of a proline between positions 2 and 1 of the δ subunit in the nominal absence of extracellular Ca 2+ or Mg 2+ (Supplementary Table 3). Unbroken lines are cubic spline interpolations. The indicated membrane boundaries are tentative. Our interpretation of the data suggests that the lumen of the pore is to the right of the plot. 9

10 a δpgdcgepk b δpgdcpgek ph 6.0 (symmetrical) δpgdcgpek δpgdcpgek Shut Sublevel Main level ph 7.4 (symmetrical) δpgdpcgek ph 9.0 (symmetrical) δpgpdcgek δppgdcgek 20 ms 10 pa 25 ms 10 pa Supplementary Figure 7. The effect of proline insertions in the M1 M2 loop is not highly position specific. a, Singlechannel inward currents (cell-attached configuration; ~ 100 mv; 1 µm ACh; solutions 2 and 3) recorded from AChRs having single prolines inserted at six different positions along the M1 M2 loop of the δ subunit. Note that, regardless of the position, all insertions reveal a proton-binding site. Importantly, the relative insensitivity of this phenomenon to the particular position occupied by the inserted proline within the muscle-achr s M1 M2 loop mirrors the similarly loose position dependence of the cation-to-anion selectivity-converting effect of these mutations on the α7 AChR 2. The traces shown correspond to currents recorded at ph pipette 7.4 with the exception of the trace at the bottom, which was recorded at ph pipette 6.0 to make the protonation events of the 0 lysine more evident. The burst-prolonging mutation was εt264p. Mutations are indicated on the M1 M2 loop sequences with underlined bold symbols denoting insertions. b, ph dependence of the mainlevel sublevel current fluctuations (outside-out configuration; 100 mv; 1 µm ACh; solutions 4 and 5) recorded from the mutant having a proline inserted between positions 4 and 2 (note that we omit the 3 label for the α1, β1 and δ subunits; see Fig. 1a). Only a section of a long burst of openings recorded at ph 9.0 is shown. 10

11 Shut States-H + H 2 O, OH, B Open-H + H 2 O, OH, B Shut States H 3 O +, H 2 O, BH Open H 3 O +, H 2 O, BH HO 2 2 OH B Deprotonation rate = k [ H O] + k [ OH ] + k [ B ] H2O 2 + BH Protonation rate = k [ H O ] + k [ H O] + k [ BH ] + + HO 3 K Deprotonation rate [H O + = ] Protonation rate a 3 pk a = logk a Supplementary Figure 8. Experimental estimation of pk a values using electrophysiological recordings. The AChR interconverts among closed, desensitized (collectively referred, here, to as shut states ) and open-channel conformations. The association and dissociation of a single proton to the open conformation is manifest as fluctuations of the single-channel current amplitude between two discrete levels, provided that the proton-binding side chain is close enough to the ionpermeation pathway to exert a noticeable electrostatic effect. We refer to the current level corresponding to the neutral open state as the main level ; to the current level corresponding to the pore with a protonated basic side chain as the sublevel ; and to that corresponding to the pore with a deprotonated acidic side chain as the superlevel. Kinetic analysis of the temporal sequence of current sojourns in the different conductance levels (see Methods) yields all the transition rates in the kinetic scheme. These include the rates of protonation and deprotonation of the ionizable side chain in question in the openchannel conformation, which, along with the ph of the membrane-bathing solution, can be combined to calculate the pk a of the engineered side chain. The three proton donors and three proton acceptors present in the solutions are indicated. BH and B denote the protonated and deprotonated forms of the H + -buffer, respectively. Note that the mean duration of sojourns in either open-channel current level depends on the kinetics of both proton transfer and channel shutting (unbroken arrows). The Schwarz criterion 54 was used to estimate the most likely number of shut states in the model. 11

12 15.0 mv 25.0 mv 35.0 mv 45.1 mv 55.1 mv 65.2 mv 75.2 mv 85.2 mv 2 ms 50 pa Supplementary Figure 9. Estimation of reversal potentials. Macroscopic-current traces recorded from the wild-type muscle AChR under KCl-dilution conditions (outside-out configuration; ph 7.4, both sides; solutions 8 and 9). Each current trace is the response of the channel to a brief pulse of 100 µm ACh. Transmembrane potentials were corrected for the presence of non-zero liquid-junction potentials. 12

13 a Wild type βv266m Shut δs268q Main level εt264p b Voltage (mv) 30 ms 10 pa Wild type βv266m δs268q εt264p Current (pa) Supplementary Figure 10. Burst-prolonging mutations. a, Single-channel inward currents (cell-attached configuration; ~ 100 mv; 1 µm ACh; ph pipette 7.4; solutions 2 and 3) recorded from AChRs having mutations that prolong the mean duration of bursts of openings. For comparison, a wild-type trace is also shown. b, Single-channel I V relationships (cell-attached configuration; 1 µm ACh; ph pipette 7.4; solutions 2 and 3) recorded from the four constructs in a. To facilitate the visual comparison of the slopes, each curve was displaced along the voltage axis so that it extrapolates exactly to the origin. The values of the single-channel conductances are: ± 0.9 ps for the wild type; ± 0.8 ps for the βv266m mutant; ± 0.8 ps for the δs268q mutant; and ± 0.5 ps for the εt264p mutant. The negligible effect of these burstprolonging mutations on single-channel conductance and charge selectivity (Fig. 1b) amply justifies their use in our experiments. 13

14 doi: /nature09229 Supplementary Table 1. Composition of solutions used for electrophysiological recordings (in terms of concentrations) Solution number Type of recording Compartment KCl (mm) NaCl (mm) CaCl 2 (mm) MgCl 2 (mm) KF (mm)* EGTA (mm) ph buffer (mm) Mannitol (mm) 1 Cell-attached (with divalent cations in Pipette the pipette) 2 Cell-attached (without divalent cations in the pipette) Pipette Cell-attached (with or without divalent cations in the pipette) 4 Outside-out (equilibrium/singlechannel recordings) 5 Outside-out (equilibrium/singlechannel recordings) 6 Outside-out (ligand-concentration jumps/kinetics) 7 Outside-out (ligand-concentration jumps/kinetics) 8 Outside-out (ligand-concentration jumps/reversal potentials) 9 Outside-out (ligand-concentration jumps/reversal potentials) Bath Pipette Bath Pipette Theta-tube barrels Pipette Theta-tube barrels *Fluoride was added to the pipette solution because it seemed to increase the stability of fast-perfused outside-out patches. This anion does not permeate appreciably through the wild-type α1 GlyR 55. The ph buffer was MES at ph 6.0 and 6.5, HEPES at ph 7.4 and TABS at ph 9.0. All solutions were titrated to final ph with KOH. This pipette solution also contained 1 µm ACh. This bath solution also contained 1 µm ACh. The solution in one of the two barrels of the theta-type tubing also contained ligand (100 µm ACh or 10 mm Gly, as indicated). 14

15 Supplementary Table 2. Some functional properties of lysine-to-alanine mutants at position 0 AChR construct Single-channel conductance (ps)* Deactivation time constant (ms) Entry-intodesensitization time constant (ms) Recovery-fromdesensitization time constant (ms) Wild type ± ± 0.06 (25) 32.6 ± 1.2 (13) 306 ± 19 (5) βk253a ± ± 0.04 (21) 18.3 ± 1.1 (27) 271 ± 20 (7) δk256a ± ± 0.06 (24) 16.4 ± 1.2 (20) 502 ± 32 (5) εk252a ± ± 0.07 (12) 15.3 ± 1.5 (15) 427 ± 17 (3) *Means and standard errors were estimated from linear fits to the I-V relationships in Supplementary Fig. 5a. Means and standard errors were calculated from the time-constant estimates obtained from a number (indicated in parentheses) of independent experiments performed as described in Methods and in Supplementary Fig. 5. Means and standard errors were estimated from mono-exponential fits to the plots in Supplementary Fig. 5d. The numbers in parentheses indicate the number of independent experiments analyzed to generate these plots. 15

16 Supplementary Table 3. Effect of M1 M2 loop mutations on the proton-transfer properties of the basic side chain at position 0 Sequence 7 0 * Extent of block Deprotonation rate (s 1 ) Protonation rate (s 1 ) pk a, pore (ph 7.3) pk a Intervals No. αptdsgpek ± αptdspgek ± ,363 ± 347 6,575 ± ± ,306 3 αptdspek ± βppdagpek ± βppdapek ± δpgdcgpek ± ,499 ± 117 4,787 ± ± ,572 6 δpgdcgaek ± ,961 ± 112 6,668 ± ± ,095 3 δpgdcgtek ± δpgdcggek ± ,778 ± 185 8,810 ± ± ,007 7 δpgdcagek ± ,344 ± 230 7,029 ± ± ,488 4 δpgdctgek ± δpgdcpgek ± ,648 ± 758 4,369 ± ± ,113,084 5 δpgdcpger ± ,776 ± 10 5,150 ± ± ,552 3 δpgdcgpak ± ,646 ± 103 3,476 ± ± ,150 3 δpgdpcgek ± δpgdcpek ± δpgdcaek ± δpgdcsek ± δpgdcnek ± εpaqagpqk ± ,151 ± ± ± ,141 5 *Underlined bold symbols denote insertions whereas bold symbols (without the underline) denote substitutions. Means and standard errors. The standard-error values were calculated from the standard errors of the sublevel and main-level conductance estimates. Rates of proton binding and unbinding were estimated from cell-attached single-channel recordings (using solutions 2 and 3; see Supplementary Table 1) at an applied potential of ~ 100 mv (negative inside the cell) and ph pipette 7.4. The exception is the δpgdcgpak mutant, in which case the potential was increased to ~ 150 mv to compensate for the reduced single-channel conductance (note that this mutant lacks the glutamate at position 16

17 1 ). Rates of proton transfer could be estimated with confidence only for a subset of the M1 M2 loop mutants studied here; in all other cases, the main-level sublevel transitions were deemed to be poorly resolved. Side-chain pk a values were calculated using a value of 7.3 for the ph (Supplementary Fig. 8), a value halfway between the ph of the extracellular compartment (~7.4) and that of the cytosol (~7.2). See Methods. pk a = pk a,pore pk a,bulk. The pk a values of the side chains of lysine and arginine in bulk water were taken as 10.4 and 12.5, respectively. Total number of idealized intervals used for the estimation of proton-transfer rates. Number of independent patch-clamp recordings used for kinetic analysis. The means and standard errors of proton-transfer rates and pk a s were calculated from the estimates obtained from these individual recordings. 17

18 Supplementary Table 4. Comparison of rates of proton transfer and pk a values of substituted lysines in the presence (values in parentheses) or absence of extracellular Ca 2+ or Mg 2+ Mutation* Deprotonation rate (s 1 ) δi239k (M1) 527 ± 19 (584 ± 29) δt1 K (M2) 388 ± 54 (413 ± 32) δl8 K (M2) 552 ± 47 (740 ± 80) δa10 K (M2) 1,514 ± 66 (1,792 ± 77) δq11 K (M2) 79 ± 5 (85 ± 7) δs12 K (M2) 324 ± 7 (443 ± 11) δf14 K (M2) 2,431 ± 267 (1,799 ± 90) Protonation rate (s 1 ) 427 ± 14 (278 ± 18) 5,130 ± 318 (4,252 ± 462) 253 ± 6 (160 ± 9) 2,632 ± 126 (2,383 ± 63) 17 ± 1 (16 ± 1) 11,214 ± 232 (13,028 ± 345) 383 ± 51 (395 ± 12) pk a, pore pk a Intervals No ± 0.03 (7.08 ± 0.04) 8.52 ± 0.09 (8.41 ± 0.08) 7.07 ± 0.05 (6.74 ± 0.05) 7.64 ± 0.04 (7.53 ± 0.01) 5.33 ± 0.04 (5.27 ± 0.02) 8.94 ± 0.01 (8.87 ± 0.01) 6.60 ± 0.03 (6.74 ± 0.03) 3.09 ( 3.32) 1.88 ( 1.99) 3.33 ( 3.66) 2.76 ( 2.87) 5.07 ( 5.13) 1.46 ( 1.53) 3.80 ( 3.66) 188, , , , , , ,480 3 *Lysine substitutions in the M2 transmembrane segment are indicated using the prime-numbering system (Supplementary Fig. 1a). The kinetics of proton transfer could only be estimated at positions where the lysine side-chain s pk a is downshifted into the range. Beyond these values, protonation-deprotonation events occurring in the ph range of our solutions are rare and exceedingly brief. Rates of proton binding and unbinding were estimated from cell-attached single-channel recordings (using solutions 2 and 3; see Supplementary Table 1) at an applied potential of ~ 100 mv and ph pipette 7.4. The exception is the δq11 K mutant, in which case the ph pipette was 6.0; the ph needed to be lowered because the εnh 2 group at this position is almost permanently deprotonated at ph 7.4. Values in the presence of 1.8 mm Ca 2+ and 1.7 mm Mg 2+ in the pipette solution (using solutions 1 and 3; see Supplementary Table 1) are indicated in parentheses (taken from refs 12, 13). Side-chain pk a values were calculated using the ph of the pipette solution (Supplementary Fig. 8). pk a = pk a,pore pk a,bulk. The pk a value of the side chain of lysine in bulk water was taken as Total number of idealized intervals used for the estimation of proton-transfer rates in the nominal absence of extracellular Ca 2+ or Mg 2+. Number of independent patch-clamp recordings used for the kinetic analysis of protonation-deprotonation events occurring in the nominal absence of extracellular Ca 2+ or Mg 2+. The means and standard errors of proton-transfer rates and pk a s were calculated from the estimates obtained from these individual recordings. 18

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