Identification of a domain involved in ATP-gated ionotropic receptor subunit assembly.
P2X receptors are ATP-gated ion channels found in a variety of tissues and cell types. Seven different subunits (P2X(1)-P2X(7)) have been molecularly cloned and are known to form homomeric, and in some cases heteromeric, channel complexes. However, the molecular determinants leading to the assembly of subunits into P2X receptors are unknown. To address this question we utilized a co-immunoprecipitation assay in which epitope-tagged deletion mutants and chimeric constructs were examined for their ability to co-associate with full-length P2X subunits. Deletion mutants of the P2X(2) receptor subunit were expressed individually and together with P2X(2) or P2X(3) receptor subunits in HEK 293 cells. Deletion of the amino terminus up to the first transmembrane domain (amino acid 28) and beyond (to amino acid 51) did not prevent subunit assembly. Analysis of the carboxyl terminus demonstrated that mutants missing the portion of the protein downstream of the second transmembrane domain could also still co-assemble. However, a mutant terminating 25 amino acids before the second transmembrane domain could not assemble with other subunits or itself, implicating the missing region of the protein in assembly. This finding was supported and extended by data utilizing a chimera strategy that indicated TMD2 is a critical determinant of P2X subunit assembly.
Torres
G E
GE
Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Egan
T M
TM
Voigt
M M
MM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion
D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D011993:Recombinant Fusion Proteins; C110590:purinoceptor P2X6; D000255:Adenosine Triphosphate