Role Of The Left Flipper Domain In The Homotrimeric Assembly And Function Of P2x Receptors
BIOPHYSICAL JOURNAL(2018)
Abstract
P2X receptors (P2XRs) are trimeric ATP-gated channels (Nicke et al. EMBO J. 17, 3016-3028, 1998) that assemble from a repertoire of seven subunits, P2X1-P2X7. ATP binding triggers the opening of an intrinsic ion channel that allows for the non-selective passage of cations along their electrochemical gradients. P2X6 subunits co-assemble with other P2XR subunits to functional heterotrimeric channels, but are incapable of forming functional homotrimers. In Xenopus laevis oocytes, we have previously found that singly expressed P2X6 subunits form random homooligomers, mostly tetramers, rather than defined homotrimers (Aschrafi et al. JMB 342, 333-343, 2004). This suggested to us that P2X6 subunits have a genuine assembly defect. A sequence alignment based on the X-ray structure of the zebrafish (zf) zfP2X4R disclosed that P2X6 subunits lack nine residues forming the so-called left flipper domain; this may compromise subunit-subunit contacts and thus explain the inability of forming functional homotrimers (Kawate et al. Nature 460, 592-599, 2009). Here, we analyzed the role of the left flipper domain in the homotrimeric assembly and ATP-gated channel function by site-directed mutagenesis, blue native polyacrylamide gel electrophoresis (BN-PAGE) and two-electrode voltage-clamp electrophysiology (TEVC). We found that the insertion of residues 281-287 or 290-297 of the rP2X1 or hP2X2 subunit, respectively, into the rP2X6 subunit neither enabled efficient homotrimerization nor ATP-gated channel function. A deletion of the appropriate left flipper residues from rP2X1, hP2X2, hP2X3 or rP2X7 did not prevent homotrimeric assembly, but interfered with the function of the P2XRs as ATP-gated ion channels. The respective left flipper deletions decelerated the desensitization of the rP2X1R and the hP2X3R, while they abolished the functions of the hP2X2R and the rP2X7R. These data will be discussed in terms of the known P2XR X-ray structures.
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Key words
receptors,left flipper domain,homotrimeric assembly
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