Contribution of Transmembrane Regions to ATP-gated P2X2 Channel Permeability Dynamics

Abstract

ATP-gated P2X 2 channels undergo activation-dependent permeability increases as they proceed from the selective I 1 state to the I 2 state that is readily permeable to organic cations. There are two main models about how permeability changes may occur. The first proposes that permeability change-competent P2X channels are clustered or redistribute to form such regions in response to ATP. The second proposes that permeability changes occur because of an intrinsic conformational change in P2X channels. In the present study we experimentally tested these views with total internal reflection fluorescence microscopy, electrophysiology, and mutational perturbation analysis. We found no evidence for clusters of P2X 2 channels within the plasma membrane or for cluster formation in response to ATP, suggesting that channel clustering is not an obligatory requirement for permeability changes. We next sought to identify determinants of putative intrinsic conformational changes in P2X 2 channels by mapping the transmembrane domain regions involved in the transition from the relatively selective I 1 state to the dilated I 2 state. Initial channel opening to the I 1 state was only weakly affected by Ala substitutions, whereas dramatic effects were observed for the higher permeability I 2 state. Ten residues appeared to perturb only the ). The data favor the hypothesis that permeability changes occur because of permissive motions at the interface between first and second transmembrane domains of neighboring subunits in pre-existing P2X 2 channels.ATP-gated P2X channels are a large family of transmittergated cation channels (1-3). P2X channels are widely expressed in the brain where they mediate, presynaptically facilitate, and postsynaptically modulate fast synaptic transmission (1, 2, 4). P2X subunits (P2X 1 -P2X 7 ) are the products of seven genes and form at least 11 heteromeric channels (5). P2X channels are likely trimeric (6 -10), and each subunit possesses two transmembrane domains, both of which are thought to contribute to the pore (11)(12)(13)(14). The cytosolic domain of the protein consists of amino and carboxyl termini. P2X channels are cation-selective (2), with some displaying high Ca 2ϩ fluxes (15). P2X 2 , P2X 4 , and P2X 7 channels also display permeability dynamics, whereby the channel pore dilates in an ATP activation-and time-dependent manner from a fairly selective I 1 state to the I 2 state that is also permeable to organic cations (16 -24). The transition from I 1 to I 2 typically takes several seconds. Permeability changes have been studied most for recombinant P2X 2 , P2X 4 , and P2X 7 channels (2, 22), but there is also evidence that permeability changes occur for natively expressed channels in cells throughout the body, including neurons (16,19,20,(25)(26)(27)(28).Activation-dependent changes in ionic selectivity for P2X channels imply that the selectivity filter may be dynamic and undergo conformational re-arrangements to switch its preference between ions (22, 29). Two different models are proposed...

Patch–clamp coordinated spectroscopy shows P2X 2 receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

“…1G). Our data for WT P2X 2 reproduce past work with P2X 2 (2,4,6,19) and allowed us to determine whether Panx-1 and CBX affected NMDG ϩ permeability. We found that the shift in NMDG ϩ E rev was identical from cells expressing WT P2X 2 , P2X 2 with Panx-1, or P2X 2 with CBX (ϩ19 Ϯ 2, ϩ20 Ϯ 2, and ϩ19 Ϯ 2 mV, respectively; n ϭ 10, 9, and 8) (Fig.…”

“…1 A). The second model posits that the I 2 state is an intrinsic property of P2X receptors involving slow conformational changes that allow organic cations to flow (2)(3)(4)(5)(6)19). We call this the ''gating model'' (Fig.…”

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

“…PCR amplification included denaturation at 96°C for 5 min, 48 cycles at 94°C for 45 s, 58°C for 45 s, and 72°C for 90 s, followed by 72°C for 10 min. Samples (10 µl) of PCR product were analyzed by methods have been described in detail previously (Eickhorst et al, 2002;Khakh and Egan, 2005).…”

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

“…Functions of the TM domains in channel gating have been deeply studied, and its roles in pore location, ion permeability and structural rearrangements were confirmed by crystal structures [88][89][90][91][92][93][94][95][96][97] . Under normal conditions, pore opening of P2X receptors is controlled by ATP binding.…”