Silberberg SD et al. (2005), Secondary structure and gating rearrangements o...

XB-ART-2105

J Gen Physiol 2005 Apr 01;1254:347-59. doi: 10.1085/jgp.200409221.

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Secondary structure and gating rearrangements of transmembrane segments in rat P2X4 receptor channels.

Silberberg SD , Chang TH , Swartz KJ .

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P2X receptors are cation selective channels that are activated by extracellular nucleotides. These channels are likely formed by three identical or related subunits, each having two transmembrane segments (TM1 and TM2). To identify regions that undergo rearrangement during gating and to probe their secondary structure, we performed tryptophan scanning mutagenesis on the two putative TMs of the rat P2X4 receptor channel. Mutant channels were expressed in Xenopus oocytes, concentration-response relationships constructed for ATP, and the EC50 estimated by fitting the Hill equation to the data. Of the 22 mutations in TM1 and 24 in TM2, all but one in TM1 and seven in TM2 result in functional channels. Interestingly, the majority of the functional mutants display an increased sensitivity to ATP, and in general these perturbations are more pronounced for TM2 when compared with TM1. For TM1 and for the outer half of TM2, the perturbations are consistent with these regions adopting alpha-helical secondary structures. In addition, the greatest perturbations in the gating equilibrium occur for mutations near the outer ends of both TM1 and TM2. Surface biotinylation experiments reveal that all the nonfunctional mutants traffic to the surface membrane at levels comparable to the WT channel, suggesting that these mutations likely disrupt ion conduction or gating. Taken together, these results suggest that the outer parts of TM1 and TM2 are helical and that they move during activation. The observation that the majority of nonconducting mutations are clustered toward the inner end of TM2 suggests a critical functional role for this region.

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Species referenced: Xenopus laevis
Genes referenced: p2rx4 p2rx7 pycard

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	Figure 1. . Putative transmembrane regions of P2X receptor channels. (A) Schematic representation of the general topology of a P2X receptor channel subunit. (B) Sequence alignment of TM1 (left) and TM2 (right) of P2X4 receptor channels from different species (top) and of rat P2X1–P2X7 (bottom). Bold residues are identical across all known P2X receptor channels (except for a Y to F substitution in Xenopus P2X7). The major findings of this study are summarized above the sequences. X indicates mutations to tryptophan that result in nonconducting channels. Empty circles and bars indicate positions and regions of large perturbations in EC50 induced by the mutations, respectively.
	Figure 2. . Generation of concentration–response relationships. (A) Representative current traces in response to ATP recorded from oocytes expressing WT P2X4 receptor channels. Each trace is from a different oocyte. A reference concentration of ATP (3 μM) was applied first followed 2 min later by a test concentration (indicated by solid bars above the current traces). Calibration bars: 10 s and 0.2 μA. (B) Superimposed normalized current traces of WT (left) and Y42W (right) receptor channels. The bars above the current traces indicate the duration of ATP application. The concentrations of ATP are 1.0, 3.0, 10, 30, and 100 μM for WT and 0.1, 0.3, 1.0, 3.0, and 10 μM for Y42W. (C) Normalized concentration–response relationships for the WT receptor channel (full circles) and for Y42W (empty squares). Each point is the average ± SEM of 4–5 measurements. The EC50 and (n) estimated from fitting the Hill equation to the WT and Y42W data are 11.8 ± 0.7 μM, (1.5 ± 0.1), and 1.4 ± 0.1 μM, (1.3 ± 0.1), respectively.
	Figure 3. . Tryptophan substitution at many positions in TM1 increases sensitivity to ATP. Estimated EC50 ± SEM (A) and fold changes in EC50 relative to WT (B) for each of the point mutations in TM1. Only V28W does not give rise to measurable currents. A concentration–response relationship could not be generated for L40W. The mean ± SEM of the EC50 for WT receptor channels is represented by a vertical line and vertical dashed lines, respectively. Every third or fourth residue exhibits a greater perturbation in EC50 relative to its neighboring residues (bold typeface, full bars). Toward the outer region of TM1 there are six successive mutations with large perturbations in EC50 (vertical bar in A).
	Figure 4. . TM1 is likely an α-helix. Helical wheel representation (A) and helical net diagram (B) of TM1. The residues in the helical wheel representation progressively decrease in size from the outer to inner ends of TM1 (residues W46–V49 are not shown). The residues labeled in bold typeface in Fig. 3 map to one face of a presumed α-helix. (C) Fourier analysis of the fold change in EC50 for the region of TM1 underlined above. The primary peak of the power spectrum occurs at 102°. The α-PI calculated from the spectrum is 2.26. The EC50 for L40W was taken to be 30 μM (see Table I).
	Figure 5. . Tryptophan substitution increases sensitivity to ATP in many positions in TM2. Estimated EC50 ± SEM (A) and fold changes in EC50 relative to WT (B) for each of the point mutations in TM2. Seven residues, six of which are toward the inner region of TM2, do not give rise to measurable currents (light gray typeface). A concentration–response relationship could not be determined for V351W. The mean ± SEM EC50 for WT is represented by a vertical line and vertical dashed lines, respectively. Toward the outer region of TM2 the perturbations in EC50 are large and the four mutants with the greatest perturbations are four residues apart. (bold typeface, full bars).
	Figure 6. . The extracellular region of TM2 seems to be an α-helix. Helical wheel representation (A) and helical net diagram (B) of TM2. The residues in the helical wheel representation progressively decrease in size from the outer to inner ends of TM2. The residues labeled in bold typeface in Fig. 5 map to one face of a presumed α-helix.
	Figure 7. . All of the nonfunctional mutants form nonconducting channels. (A) Western blots from SDS-PAGE of P2X4 receptor channels obtained from surface membrane preparation (top) and total crude membrane preparation (bottom). Each lane contains the protein from an equivalent of 20 oocytes (surface) or 1 oocyte (total). All the oocytes expressing mutant channels were treated with biotin. The four left lanes and the eight right lanes are from two separate experiments. Numbers to the left are molecular weight markers in kD. (B) Western blots from SDS-PAGE of c-myc–tagged Shaker I477D and F481W protein obtained from surface membrane preparation (right two lanes) and total crude membrane preparation (left two lanes). Each lane contains the protein from an equivalent of 20 oocytes (surface) or 1 oocyte (total). F481W protein has two dominant forms, a core-glycosylated form (∼70 kD) and a mature form (∼100 kD). I477D exhibits only the core-glycosylated form. Numbers to the left are molecular weight markers in kD. (C) Mean ± SEM currents (averaged from 10 oocytes) from oocytes coinjected with 1:1 mixtures of cRNA for the WT and nonconducting mutant channel subunits. Oocytes injected with a 1:1 dilution of WT cRNA served as a control. The dashed line represents the expected current if the subunits randomly assemble to form trimers and a single mutant subunit prevents channel function. Significant differences between the control and each of the other groups was assessed with ungrouped Student's t test. , P < 0.05; *, P < 0.001.
	Figure 8. . Proposed secondary structure and packing of the TMs of P2X4 receptor channels. (A) TM1 and the outer region of TM2 are depicted as α-helical structures. The residues that exhibit the greatest perturbations in EC50 relative to the neighboring residues are in red. The regions of large perturbation in EC50 are shaded gray. Mutations that result in nonconducting channels are crossed out. (B) A schematic representation of the proposed packing of the TMs in the outer and inner regions of the membrane. Toward the inside, TM1 is depicted as being more peripheral, interacting with TM2 through the residues labeled in A in red. The inner triangle denotes that the secondary structural for the inner region of TM2 is undefined. Toward the outside, both TM1 and TM2 are depicted as α-helices, and are likely more tightly packed.

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