Control of P2X2 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 I1 state to the I2 state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X2 (rP2X2) channels does not occur at mouse P2X2 (mP2X2) 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 rP2X2 and mP2X2 channels have a pore diameter of ∼11 Å in the I1 state, but the transition to the I2 state increases the rP2X2 diameter by at least 3 Å. The I1 to I2 transition occurs with a rate constant of ∼0.5 s−1. The data focus attention on specific residues of P2X2 channel cytoplasmic domains as determinants of permeation in a state-specific manner.

Desensitization Masks Nanomolar Potency of ATP for the P2X1 Receptor

“…Moreover, a chimera containing the extracellular domain of the rP2X 3 receptor flanked by the transmembrane and cytosolic domains of the rP2X 2A receptor exhibits a 30-fold lower EC 50 value compared with the rP2X 2A receptor (26). A leftward shift of the concentration-response curve is also observed when the native rP2X 2A ectodomain is substituted with the rP2X 4 ectodomain, suggesting that transmembrane domain-flanking sequences can also affect agonist potency (27).…”

“…Seven genes encoding P2X subunits (P2X [1][2][3][4][5][6][7] ) are known in mammals. All P2X subunits share a common membrane topology, with two hydrophobic membrane-spanning segments (M1 and M2) separated by a large extracellular loop of ϳ300 amino acid residues, which comprise the ATP-binding domain.…”

“…All P2X subunits share a common membrane topology, with two hydrophobic membrane-spanning segments (M1 and M2) separated by a large extracellular loop of ϳ300 amino acid residues, which comprise the ATP-binding domain. At least part of the pore of the P2X receptor is lined by M2 (2), which includes a conserved glycine residue that is likely to constitute the channel gate (3), but also residues of the Cterminal cytoplasmic tail appear to play a role in controlling channel permeability (4). The cytoplasmic N-terminal tails of the various subunit isoforms are approximately the same length (ϳ30 amino acid residues), whereas the cytoplasmic C-terminal tails are highly variable in length.…”

“…Based on their sensitivity to the ATP analog ␣,␤-MeATP 1 and the rate of current desensitization, homomeric P2X receptors are generally divided into at least two categories: rapidly desensitizing (P2X 1 and P2X 3 ) and slowly or non-desensitizing (P2X 2A , P2X 4 , and P2X 7 ) receptors. The term P2X 2A distinguishes the nondesensitizing full-length P2X 2A subunit from the desensitizing C-terminal splice variant termed P2X 2B , which lacks a stretch of 69 amino acids C-terminal to M2 (8).…”

P2X7 Receptor Cell Surface Expression and Cytolytic Pore Formation Are Regulated by a Distal C-terminal Region

“…The fact that these receptors are able to form pores but do not contain the P2X7 pore-enabling domain (residues 551-581) indicates that a separate P2X pore-forming domain may exist. Khakh and Lester (7) proposed that the change between channel and pore states was achieved through a conformational change within the selectivity filter and have recently reported that the permeation of the I 2 state of the P2X2R is modulated by specific residues (432 and 444) within the C-terminal tail (26). The truncated P2X2 a R-(1-403) was reported to be permeable to large cations (positively charged N-methyl-D-glucamine) (25), which is in contrast to the minimum length requirement of 582 residues for the P2X7R to form pores seen in the present study.…”