Kinetics of Conformational Changes Revealed by Voltage-Clamp Fluorometry Give Insight to Desensitization at ATP-Gated Human P2X1 Receptors

Abstract

ATP acts as an extracellular signaling molecule at cell-surface P2X receptors, mediating a variety of important physiologic and pathophysiologic roles. Homomeric P2X1 receptors open on binding ATP and then transition to an ATP-bound closed, desensitized state that requires an agonist-free washout period to recover. Voltage-clamp fluorometry was used to record ion channel activity and conformational changes simultaneously at defined positions in the extracellular loop of the human P2X1 receptor during not only agonist binding and desensitization but also during recovery. ATP evoked distinct conformational changes adjacent to the agonist binding pocket in response to channel activation and desensitization. The speed of recovery of the conformational change on agonist washout was statedependent, with a faster time constant from the open (5 seconds) compared with the desensitized (75 seconds) form of the channel. The ability of ATP to evoke channel activity on washout after desensitization was not dependent on the degree of conformational rearrangement in the extracellular loop, and desensitization was faster from the partially recovered state. An intracellular mutation in the carboxyl terminus that slowed recovery of P2X1 receptor currents (7-fold less recovery at 30 seconds) had no effect on the time course of the extracellular conformational rearrangements. This study highlights that the intracellular portion of the receptor can regulate recovery and shows for the first time that this is by a mechanism independent of changes in the extracellular domain, suggesting the existence of a distinct desensitization gate in this novel class of ligand gated ion channels.

X-ray structures define human P2X3 receptor gating cycle and antagonist action

“…Proposed mechanisms are similar to the “hinged lid” or “ball and chain” models described for voltage-gated sodium and shaker potassium channels, respectively, with a distinct but unidentified desensitization gate 21,26 . To date, there are no structures of a P2X receptor in the desensitized state and currently available structures of the zebra fish P2X 4 receptor (zfP2X 4 ) in apo and open state conformations do not visualize cytoplasmic residues 27–29 .…”

Role of domain calcium in purinergic P2X2 receptor channel desensitization

“…The NH 2 -terminal residues also participate in P2XR desensitization; the threonine-18 residue has been reported to be important for P2X2R desensitization (2), and the hydrophobic serine-15 residues have been reported for P2X3R desensitization (17). In the P2X1R, the contribution of both NH 2 and COOH termini to receptor desensitization domains has been found with the use of chimeric receptors and voltage-clamp fluorometry (1,16). Thus desensitization of P2XRs is a complex process that depends on multiple domains and probably involves the interaction of those domains and conformational changes that occur during the gating process.…”

Investigating ion channel conformational changes using voltage clamp fluorometry

“…Seven P2X receptor isoforms are known (P2X1 e 7). The recent crystal structures of zebrafish P2X4 receptors in closed and open state have provided key insights into ATP binding and receptor gating mechanisms (Hattori and Gouaux, 2012), and the conformational changes governing channel opening and kinetics of desensitization have recently been addressed by VCF (Fryatt and Evans, 2014;L€ orinczi et al, 2012). P2X receptors are formed by three subunits and each subunit consists of an extracellular loop domain and two a-helical TM segments that are terminated by intracellular N-and C-termini (Fig.…”

“…These results confirmed the ATP binding site as proposed by crystallography studies and provided new insights into the proposed ATP binding orientation (Chataigneau et al, 2013;L€ orinczi et al, 2012). Recently, another VCF study on P2X1 receptors investigated the relationship between conformation rearrangements in the extracellular loop and recovery of the channel from desensitization (Fryatt and Evans, 2014). Monitoring fluorescence at the methanethiosulphonate-5(6)-carboxytetramethylrhodamine (MTS-TAMRA; MW ¼ 567.6) labelled K190C residue, they showed that the recovery rate of the fluorescence signal on agonist washout was faster from the open relative to the desensitized channel state.…”

“…Furthermore, an intracellular mutation in the carboxyl terminus known to slow the recovery from desensitization had no effect on the time-course of the extracellular conformational rearrangements. This shows that an intracellular domain can regulate recovery from desensitization, independent of changes in the extracellular domain, implying the existence of a distinct desensitization gate (Fryatt and Evans, 2014).…”