Control of P2X(2) channel permeability by the cytosolic domain

Abstract

ATP-gated P2X channels are the simplest of the three families of transmitter-gated ion channels. Some P2X channels display a time- and activation-dependent change in permeability as they undergo the transition from the relatively Na(+)-selective I(1) state to the I(2) state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X(2) (rP2X(2)) channels does not occur at mouse P2X(2) (mP2X(2)) channels expressed in oocytes. Domain swaps, species chimeras, and point mutations were employed to determine that two specific amino acid residues in the cytosolic tail domain govern this difference in behavior between the two orthologous channels. The change in pore diameter was characterized using reversal potential measurements and excluded field theory for several organic ions; both rP2X(2) and mP2X(2) channels have a pore diameter of approximately 11 A in the I(1) state, but the transition to the I(2) state increases the rP2X(2) diameter by at least 3 A. The I(1) to I(2) transition occurs with a rate constant of approximately 0.5 s(-1). The data focus attention on specific residues of P2X(2) channel cytoplasmic domains as determinants of permeation in a state-specific manner.

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 (R_x = radius; see materials and methods) in order of increasing size are: dimethylammonium (2.7 Å), 2-(methyl-amino)-ethanol (3.1 Å), Tris⁺ (3.7 Å), and NMDG⁺ (4.5 Å), and their mobilities in free solution are 51.8, 33.4, 29.4, 24.3, and 50.1 10⁻⁴m²S mol⁻¹, for dimethylammonium, 2-(methyl-amino)-ethanol, Tris⁺, NMDG⁺, and Na⁺.

Figure 1.

Properties of permeability changes at rP2X₂ channels. (A) Representative current waveform from a voltage-clamped oocyte (−60 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 I_{Na ⁺}, the statistics for which are presented in Table I. (B) I-V relations determined at the peak of I₁, I₂, and at I_{Na ⁺}: 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).

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 (−60 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.

Figure 3.

rP2X₂ and mP2X₂ channels differ with respect to permeability changes. (A) rP2X₂: steady-state current waveform (−60 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.

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.

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 P_NMDG+/P_{Na ⁺} and (B) I₂ P_NMDG+/P_{Na ⁺}. 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.

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 P_NMDG+/P_{Na ⁺} for the I₂ and I₁ states. Note that I₁ P_NMDG+/P_{Na ⁺} is constant for all channels, but I₂ P_NMDG+/P_{Na ⁺} 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.

Figure 8.

Relationship of permeability changes to steady-state currents, I₁ and I₂. (A) The relationship between I₁ amplitude and I₂ P_NMDG+/P_{Na ⁺}. (B) The relationship between I₁ P_NMDG+/P_{Na ⁺} and I₂ P_NMDG+/P_{Na ⁺}. (C) The relationship between I₂ amplitude and I₂ P_NMDG+/P_{Na ⁺}; 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.

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 “nonpermissive state” 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).