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Figure 4. . Pore sizes determined using EFT. Top and bottom panels show graphs used to determine the narrowest part of the channel pore for rP2X2 and mP2X2 channels, respectively, in I1 (within 1s of applying ATP) and I2 states (up to 30 s after applying ATP). The lines are linear regressions except for rP2X2 I2, which is a cubic spline. Please refer to materials and methods for details of the pore size calculations. The ions (Rx = radius; see materials and methods) in order of increasing size are: dimethylammonium (2.7 Ã
), 2-(methyl-amino)-ethanol (3.1 Ã
), Tris+ (3.7 Ã
), and NMDG+ (4.5 Ã
), and their mobilities in free solution are 51.8, 33.4, 29.4, 24.3, and 50.1 10â4m2S molâ1, for dimethylammonium, 2-(methyl-amino)-ethanol, Tris+, NMDG+, and Na+.
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Figure 1. . Properties of permeability changes at rP2X2 channels. (A) Representative current waveform from a voltage-clamped oocyte (â60 mV). The cell was bathed in an NMDG+-containing extracellular solution and then at the indicated time ATP was applied, first with NMDG+ and then with Na+ in the extracellular solution. The record illustrates the three current phases, I1, I2, and INa+, the statistics for which are presented in Table I. (B) I-V relations determined at the peak of I1, I2, and at INa+: note that the reversal potentials differ. The ATP-evoked current reversal potential does not shift over time in extracellular solutions that contain only Na+ (Khakh et al., 1999).
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Figure 2. . Basic properties of rP2X2 and mP2X2 channels. (A) Concentration-effect curves for ATP at rP2X2 and mP2X2. There is a significant difference in EC50. (B) I-V relations from voltage steps (â60 to 60 mV in 10-mV steps) for mP2X2 and rP2X2. Note that rectification is approximately equal for both channels, but that there is greater variability in the data for outward currents as compared with inward current. A similar trend for natively and heterologously expressed channels has been reported previously (Khakh et al., 1995; Evans et al., 1996). In this and all other figures the error bars are omitted when they are smaller than the symbols used.
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Figure 3. . rP2X2 and mP2X2 channels differ with respect to permeability changes. (A) rP2X2: steady-state current waveform (â60 mV) in NMDG+ extracellular solutions. ATP was applied when indicated and evoked an outward current that became inward over time. (B) Reversal potentials at the peak of I1 and I2 for rP2X2. (C) mP2X2: steady-state current waveform in NMDG+ extracellular solutions. ATP was applied for the period indicated and evoked an outward current that waned to a steady-state level. (D) Reversal potentials at the peak of I1 and I2 for mP2X2.
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Figure 5. . Comparison of rP2X2 and mP2X2 sequences. The top panel shows an alignment for the entire rP2X2 and mP2X2 protein sequences (black lines are above predicted transmembrane domains and the black dots indicate the conserved cysteine residues), whereas the middle panel shows a representation of P2X subunit topology. The bottom panel shows an alignment of the mP2X2 cDNA used in this study, and the corresponding sequence of the mP2X2 gene. The codons for the amino acids that differ between mP2X2 and rP2X2 are shown in gray. Note that the genomic sequence agrees with the cDNA sequence in each case, assuring that the mP2X2 cDNA contains no PCR errors. The boxed regions correspond to identical sequence.
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Figure 6. . No single amino acid residue in the COOH-terminal domain accounts for the differences between mP2X2 and rP2X2 channels. The scatter graphs show data for wt mP2X2, rP2X2, mEL/rCT, rEL/mCT, and 14 single point mutant channels: (A) I1 PNMDG+/PNa+ and (B) I2 PNMDG+/PNa+. ATP-evoked currents for all mutants, chimeric, and wild-type channels were similar in amplitude for recordings in Na+ solutions, indicating approximately equivalent membrane expression.
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Figure 7. . Identification of C tail domain residues that govern permeability changes in the P2X2 channel. The top panel illustrates the location of P2X2 channel mutants, and chimeras with respect to the two TM domains. The bottom panel shows PNMDG+/PNa+ for the I2 and I1 states. Note that I1 PNMDG+/PNa+ is constant for all channels, but I2 PNMDG+/PNa+ varies depending on the composition of the C tail domain. Thus rP2X2 channels undergo large changes between I1 and I2, whereas mP2X2 channels do not. The differences can be reversed by transplanting the C tail domains (mEL/rCT and rEL/mCT) and by making chimeras that include the two most COOH-terminal rP2X2 residues of the seven that differ in this domain (see Fig. 5). The gray arrows point to uninformative or outlier constructs.
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Figure 8. . Relationship of permeability changes to steady-state currents, I1 and I2. (A) The relationship between I1 amplitude and I2 PNMDG+/PNa+. (B) The relationship between I1 PNMDG+/PNa+ and I2 PNMDG+/PNa+. (C) The relationship between I2 amplitude and I2 PNMDG+/PNa+; the two parameters are well correlated (r = 0.9). We verified that slope conductance values followed a similar trend to current peak amplitudes for the majority of mutants where such data was available. For this figure, the single point mutants shown in Fig. 6 have been pooled, and there is no point for the r414m chimers because there was no measurable I2.
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Figure 9. . A summary of rP2X2 permeability changes. The cartoon shows rP2X2 channels in closed and open states of differing diameter across the narrowest region. The narrowest region is shown as the gate. In the Closed and Open 1 state the C tail domain is diagrammatically shown as in a ânonpermissive stateâ with respect to permeability changes. The present paper shows that a conformational change occurs in the tail as, or before, the pore dilates, and this is hypothetically shown as a motion of the C tail domain away from the inner aspect of the channel, as in MscL channel gating (Sukharev et al., 2001). The model implies the existence of a state where the tail has undergone a conformation change but the pore has not yet dilated. A direct transition from Closed to Open 2 was demonstrated previously for mutant channels (Khakh et al., 1999; Virginio et al., 1999b).
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