Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors)

Upper Vestibule.

We examined the central pathway by placing cysteines at sites in the upper vestibule where the diameter of the channel is narrow (Fig. 2D). The first site represents the outermost entrance to the central pathway; it is obstructed in the closed state model of hP2X4R, in part because the side chain of Leu³⁰³ caps the opening. The entrance must open for current to flow, and we hypothesized that modification by MTSET⁺ of L303C would plug the open channel and reduce ATP-gated current if the central pathway formed a conducting pore. However, we saw no effect of coapplying ATP and MTSET⁺ on L303C currents (Fig. 2D), and no effect of MTSET⁺ on the neighboring D302C and A304C residues (<5% change; n = 5). We then moved inward, and placed cysteines near the top (A297C; pore diameter ≈ 10 Å) and bottom (P92C and N93C; pore diameter ≈ 5 Å; Fig. S3) of the upper vestibule, and again saw no effect (<5% change; n = 5) of MTSET⁺ (for examples, see Fig. 2D). The bulkier (≈8 × 16 × 16 Å) Texas Red-2-sulfonamidoethyl MTS (MTSTR) was also without effect at all of the aforementioned residues, despite the fact that it irreversibly blocks the current of the S341C transmembrane mutant (Fig. S2 C and E).

Next, we probed the narrow constriction (≈2.3 Å) between the upper and central vestibules of the zfP2X4.1R formed in part by three residues (Gln⁹⁷, Glu⁹⁸, Asp⁹⁹), including Glu⁹⁸ that coordinates Gd³⁺ just above the entrance to the central vestibule (8). We placed cysteines at the equivalent sites of the hP2 X 4R (Gln⁹⁴, Glu⁹⁵, Glu⁹⁶; Fig. S3) and failed to measure an effect of MTSET⁺ (Fig. 2 D and E) or MTSTR. Although it is possible that MTSET⁺ and MTSTR modify residues in the constriction without changing current amplitude (14), the comparative dimensions of the MTS head groups and the diameter of the pathway (8) render this highly unlikely. Another possibility is that residues in the constriction are inaccessible to MTSET⁺ and MTSTR, either because the central pathway does not form a part of the ion channel pore, or because large MTS reagents are impermeable. Although the latter option seems unlikely because of the demonstrated accessibility of the downstream S341C mutant, we nevertheless tested this possibility by using Cd²⁺, a small metal element that binds to thiol side chains exposed to water, as a permeable probe (19). We expected to measure a change in the amplitude of the ATP-gated current if the coordinated ion occludes the channel or preferentially locks it in the open or closed state, but we found that Cd²⁺ (20 μM) failed to modify (<5% change; n = 6–7) the ATP-evoked currents of any of these three hP2X4R mutants. When considered in concert with the A93C/A297C/L303C experiments, our results argue against the suggestion that the central pathway forms a major conduit for ion conduction in the hP2X4R (8, 9).

Lateral Portals.

We then tested the alternative hypothesis that cations enter the transmembrane pore through the lateral portals. We started by studying two acidic residues in the lateral portals of the hP2X4R, Glu⁵⁶ and Asp⁵⁸ (Fig. 3A), that are common to most (P2X2-P2X7) but not all (P2X1) human P2X receptors, and that are thought to concentrate cations in the extracellular vestibule of the zfP2X4.1R pore (8). Fig. 3 shows the effect of coapplying MTSET⁺ and ATP. The results were qualitatively similar in both mutants: MTSET⁺ caused irreversible reductions in the amplitudes of the ATP-gated currents. In cells expressing the E56C mutant, MTSET⁺ reduced current by 58 ± 4% (n = 9), with an apparent modification rate (K_on) of 7,057 ± 421 M⁻¹⋅s⁻¹ (Fig. 3B). By comparison, MTSET⁺ caused a slower (K_on = 3,545 ± 577 M⁻¹⋅s⁻¹) but more complete block (95 ± 1%, n = 8) of the D58C mutant (Fig. 3C). The faster block of E56C may reflect the fact that the substituted cysteine resides on the outer surface of the portal where it is more accessible to MTSET⁺ than D58C (Fig. S3).

Although Glu⁵⁶ and Asp⁵⁸ are positioned in the lateral portals/extracellular vestibule, they also lie at the lowest point of the central pathway. Therefore, it remained possible that the MTSET⁺-sensitive currents move through the central pathway before reacting with the engineered thiolates. We addressed this possibility.

First, we measured MTSET⁺ reactivity in the absence of ATP. The central pathway contains several narrow constrictions that must open to allow MTSET⁺ to access Glu⁵⁶ and Asp⁵⁸ (8). In contrast, there is no barrier to current flow in the portals that would prevent modification of the closed states of E56C and D58C mutants. We established baseline responses to ATP (100 μM), and then applied MTSET⁺ for 2–60 s in the absence of ATP. In all cases, MTSET⁺ caused significant and persistent inhibitions of currents elicited by subsequent applications of ATP (Fig. 3 D and E). In some cases, the ATP-gated currents showed a partial recovery in the first 3 min after washout of MTSET⁺. A similar effect is reported for MTSET⁺ inhibition of cysteine-substituted rat P2X4Rs, which is thought to reflect recycling of unmodified receptors to the plasma membrane (20) (see also ref. 7). However, in no case did the amplitude of the ATP currents return to their control values, and a significant inhibition remained even after a 15-min wash (Fig. 3 D–F). Further, the persistent inhibition of ATP-gated current by MTSET⁺ was immediately and fully reversed by the reducing agent, DTT (1 mM; 60 s) (Fig. 3 D–F). The same protocol had no effect on the wt hP2X4R (Fig. 2C), or on mutant receptors containing cysteine substitutions in the narrowest constriction of the central pathway (Fig. 2E), suggesting that the majority of the plasmalemmal E56C and D58C receptors were covalently modified by MTSET⁺.

Second, we measured the ability of an anionic MTS reagent to modify E56C and D58C. The profoundly acidic surface of the central vestibule of the zfP2X4.1R provides a long-range negative electrostatic environment that favors the passage of cations over anions (8, 13). The hP2X4R shows a modest Cl⁻ permeability (21), and we find that the anionic thiol-reactive drug, (2-sulfonatoethyl) MTS⁻ (MTSES⁻), causes a fast (8,348 ± 867 M^-1⋅s⁻¹) and near complete block of the S341C mutant (Fig. S2 D and E), as previously shown for the homologous residue (Thr³³⁶) of the rP2X2R (11). Like MTSET⁺, MTSES⁻ must pass through either the central pathway or the lateral portals before reaching S341C. Therefore, we coapplied MTSES⁻ (1 mM) and ATP (100 μM) to measure accessibility and found that it modified both E56C and D58C mutant receptors. MTSES⁻ caused a small (34%) but significant (P = 0.0003) potentiation of current through hP2X4R-E56C and a near complete block of ATP-gated current through hP2X4-D58C (Fig. S4 A–C). In cysteine substituted accessibility experiments, potentiation and block are both equally insightful and interpreted to indicate side chain accessibility (14). Surprisingly, the apparent modification rate at position D58C was faster for MTSES⁻ (K_on = 10,681 ± 1,123 M^-1⋅s⁻¹, n = 7) than for MTSET⁺ (≈3,500 M⁻¹⋅s⁻¹). We do not know why the anionic drug reacts quicker. Nonetheless, the fast reaction rate suggests that MTSES⁻ has an unimpeded route of access to the thiol side chain of D58C, a situation that favors the short and less charged environment of the lateral portals over the longer and highly electronegative central pathway.

Finally, we measured the effect of MTSET⁺ and MTSES⁻ on T57C because it sits between the two acidic amino acids of the lateral portals. Coapplication of MTSET⁺ caused a moderate (27 ± 5%; n = 5) but significant (P = 0.0004) inhibition of the ATP-gated current of hP2X4R-T57C (Fig. S4D). In contrast, MTSES⁻ had no affect. However, an initial application of MTSES⁻ prevented the inhibition of ATP-gated current by a subsequent application of MTSET⁺ (Fig. S4E), showing that the anionic reagent modifies the cysteine at position Tyr⁵⁷.

Studies of Single Channel Current.

The block of the ATP-gated currents of hP2X4R-E56C and hP2X4-D58C by MTSET⁺ reflects either a change in conductance or a change in gating (14). Although both effects signal accessibility, a change in conductance is the more direct demonstration that the modified side-chain lines the permeation pathway (14, 22, 23). To measure a change in conductance, we recorded ATP-gated single channel currents from the membrane patches of HEK293 cells voltage-clamped at −120 mV. The hP2X4R shows a fast and progressive loss of single channel activity in excised patches that severely limits before and after comparisons of drug effects (24). Rundown also occurs in cell-attached patches, but at a slower rate that allows repeated measurements of single channel current amplitude (Fig. S5A; ref. 24). Thus, we used the cell-attached mode to compare channel amplitudes from populations of cells bathed in a physiological salt solution with or without 1 mM MTSET⁺ before seal formation (SI Methods).

We recorded bursts of single-channel current from wt (Fig. S5B) and mutant hP2X4Rs (Fig. 4A) in the presence of ATP (0.3–1 μM) that were never seen in the absence of agonist or in untransfected cells. We found no difference in the conductances of the wt hP2X4R and the E56C single channel currents that measured 17.8 ± 0.8 pS (n = 7) and 16.2 ± 0.4 pS (n = 23), respectively. In contrast, the conductance of the D58C mutant (21.7 ± 0.6, n = 26) was significantly larger than that of the other two (P < 0.01), providing the first clue that D58C lines the permeation pathway. We then compared the average current amplitudes of the two cysteine-substituted receptors expressed in HEK293 cells bathed for 3 min in 1 mM MTSET⁺ and saw significant (P < 0.0001) reductions (≈15% for E56C and ≈40% for D58C) in the size of the single channel currents (Fig. 4B). The fact that cysteine modification decreases conductance provides robust support for the model that E56C and D58C line the permeation pathway and, thus, validates the hypothesis that the lateral portals are a primary entrance to the transmembrane pore.

The ATP-gated whole-cell current of the hP2X4R-E56C is potentiated by coapplication of MTSES⁻ (Fig. S4A). To determine whether this potentiation reflects a change in conduction or gating, we measured single channel current after anionic thiolation and found a significant (P < 0.0001) increase (≈24%) in the size of the single channel current compared with control (Fig. 4 A and B). The observation that MTSET⁺ and MTSES⁻ have opposite effects suggests a role for electrostatics in the effect of thiolation on ATP-gated current at this position in the lateral portals.

No Effect of Removing the Fixed Negative Charge of Glu⁵⁶, Asp⁵⁸, and Glu⁹⁵ on Ca²⁺ Current.

In light of the apparent role of electrostatics described above, we next tested the hypothesis put forth by Kawate et al. that the fixed negative charge of Glu⁵⁶ and Asp⁵⁸ creates a long-range electrostatic potential that concentrates cations and repels anions near the entrance to the transmembrane pore (8). Ca²⁺ carries twice the charge of Na⁺ and is more sensitive to local changes in electrostatic fields. Thus, we measured the fraction of the total ATP-gated current carried by Ca²⁺, called the Pf%, by using patch clamp photometry as described (21, 25). We constructed single (E56Q, D58N) and double (E56Q/D58N) hP2X4R mutants with reduced charge and found that the Pf% was unchanged (Fig. S6). We therefore conclude that neither Glu⁵⁶ nor Asp⁵⁸ make a significant contribution to cation or anion selection in the hP2X4R, despite the fact that they lie in the permeation pathway. This result is consistent with the fact that the negative charges at these positions are not absolutely conserved among cationic P2X receptors. In a similar vein, we measured no effect of removing the carboxyl side chain of Glu⁹⁵ of the central pathway on the Pf% of the hP2X4R-E95C mutant (Fig. S6 D and E), despite the fact that this site forms a cation-binding site in the zfP2X4.1R (8).

Our finding that removing the fixed negative charge of Glu⁵⁶ has no effect on Pf% is interesting in light of the opposite effects of MTSET⁺ and MTSES⁻ on the single channel conductance of the E56C mutant (Fig. 4). Glu⁵⁶ is on the outer rim of the lateral portal where the negative charge of the native carboxylate may be poorly positioned to alter current (Fig. S3). In contrast, the addition of the bulky (≈6 Å) anionic headgroup of MTSES⁻ to the cysteine mutant may place fixed negative charge closer to the path of ion flow and, thus, increase single channel current by attracting cations to the lateral portals.

Central Vestibule.

We then extended our cysteine scan to determine the accessibility of four sites (Ser⁵⁹, Val⁶¹, Ser⁶², Asn⁹⁷) in the central vestibule to a range of MTS reagents (Fig. S3). Our rationale for doing this experiment was as follows: The central vestibule sits just above the lateral portals, and although most cations that exit the portals probably move passively down the transmembrane electrochemical gradient and into the cell, some may be attracted to the strong electronegative pull of acidic amino acids in the central vestibule (8, 9). In keeping with this hypothesis, we found that MTSET⁺ significantly potentiated the ATP-gated currents of S59C, S62C, and N97C mutants (Fig. S7). In contrast, MTSES⁻ potentiated the current of the receptors carrying the S59C mutation that sits just above the lateral portals, but neither altered ATP-gated current nor masked the effects of MTSET⁺ on S62C and N97C. Neither MTSET⁺ nor MTSES⁻ affected V61C. Thus, our results support the hypothesis of Kawate et al. that the negative electrostatic environment of the central vestibule attracts cations and repels cations (8). The head groups of MTSET⁺ and MTSES⁻ are approximately the same size (14), which suggests that the lack of effect of MTSES⁻ on S62C and N97C cannot be explained by bulk exclusion. Rather, the data imply that the strong electronegative environment of the central cavity impedes the flow of anions through the middle third of the central pathway, despite the fact that MTSES⁻ readily modifies downstream engineered cysteines in the lateral portals (E56C, D58C) and transmembrane pore (S341C).

Effect of Ivermectin on Thiolation.

Ivermectin (IVM) increases single channel conductance and lengthens mean open time of P2X4 receptors (7, 24, 26), at least in part through intercalation with the transmembrane helices (27). To determine whether the lateral portals lie in the allosteric pathway, we assessed the effects of MTSET⁺ on the E56C and D58C mutants before and after 5-min preincubations in 1–10 μM IVM (Fig. 5A). IVM changed the effect of MTSET⁺ in two ways. First, the rates of reaction were significantly slower (K_on values after 10 μM IVM were 1,780 ± 387 (n = 5) and 1,084 ± 200 (n = 8) for E56C and D58C, respectively). Second, the inhibitions by MTSET⁺ were significantly smaller (Fig. 5B). These findings are supported by experiments using the smaller Cd²⁺ as modifier. We found that Cd²⁺ caused a pronounced potentiation and inhibition of the ATP-gated currents of the E56C and D58C mutants, respectively, and that both effects of Cd²⁺ were absent after incubation with IVM (Fig. 5 C–E). We interpret the data with MTSET⁺ and Cd²⁺ to indicate that IVM changed the shape of the portals through an allosteric mechanism and, hence, the nature of the thiol modification.