scholarly journals Organization of ATP-gated P2X1 receptor intracellular termini in apo and desensitized states

2019 ◽  
Vol 151 (2) ◽  
pp. 146-155 ◽  
Author(s):  
Alistair G. Fryatt ◽  
Sudad Dayl ◽  
Anastasios Stavrou ◽  
Ralf Schmid ◽  
Richard J. Evans

The human P2X1 receptor (hP2X1R) is a trimeric ligand-gated ion channel opened by extracellular ATP. The intracellular amino and carboxyl termini play significant roles in determining the time-course and regulation of channel gating—for example, the C terminus regulates recovery from the desensitized state following agonist washout. This suggests that the intracellular regions of the channel have distinct structural features. Studies on the hP2X3R have shown that the intracellular regions associate to form a cytoplasmic cap in the open state of the channel. However, intracellular features could not be resolved in the agonist-free apo and ATP-bound desensitized structures. Here we investigate the organization of the intracellular regions of hP2X1R in the apo and ATP-bound desensitized states following expression in HEK293 cells. We couple cysteine scanning mutagenesis of residues R25-G30 and H355-R360 with the use of bi-functional cysteine reactive cross-linking compounds of different lengths (MTS-2-MTS, BMB, and BM(PEG)2), which we use as molecular calipers. If two cysteine residues come into close proximity, we predict they will be cross-linked and result in ∼66% of the receptor subunits running on a Western blot as dimers. In the control construct (C349A) that removed the free cysteine C349, and some cysteine-containing mutants, cross-linker treatment does not result in dimerization. However, we detect efficient dimerization for R25C, G30C, P358C, K359C, and R360C. This selective pattern indicates that there is structural organization to these regions in the apo and desensitized states in a native membrane environment. The existence of such precap (apo) and postcap (desensitized) organization of the intracellular domains would facilitate efficient gating of the channel.

2003 ◽  
Vol 376 (3) ◽  
pp. 633-644 ◽  
Author(s):  
Steven C. KING ◽  
Lisa BROWN-ISTVAN

The Escherichia coli GABA (γ-aminobutyric acid) permease, GabP, and other members of the APC (amine/polyamine/choline) transporter superfamily share a CAR (consensus amphipathic region) that probably contributes to solute translocation. If true, then the CAR should contain structural features that act as determinants of substrate specificity (kcat/Km). In order to address this question, we have developed a novel, expression-independent TSR (transport specificity ratio) analysis, and applied it to a series of 69 cysteine-scanning (single-cysteine) variants. The results indicate that GabP has multiple specificity determinants (i.e. residues at which an amino acid substitution substantially perturbs the TSR). Specificity determinants were found: (i) on a hydrophobic surface of the CAR (from Leu-267 to Ala-285), (ii) on a hydrophilic surface of the CAR (from Ser-299 to Arg-318), and (iii) in a cytoplasmic loop (His-233) between transmembrane segments 6 and 7. Overall, these observations show that (i) structural features within the CAR have a role in substrate discrimination (as might be anticipated for a transport conduit) and, interestingly, (ii) the substrate discrimination task is shared among specificity determinants that appear too widely dispersed across the GabP molecule to be in simultaneous contact with the substrates. We conclude that GabP exhibits behaviour consistent with a broadly applicable specificity delocalization principle, which is demonstrated to follow naturally from the classical notion that translocation occurs synchronously with conformational transitions that change the chemical potential of the bound ligand [Tanford (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2882–2884].


2011 ◽  
Vol 286 (33) ◽  
pp. 29207-29217 ◽  
Author(s):  
Rebecca C. Allsopp ◽  
Sam El Ajouz ◽  
Ralf Schmid ◽  
Richard J. Evans

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1166 ◽  
Author(s):  
Olivia R. Buonarati ◽  
Peter B. Henderson ◽  
Geoffrey G. Murphy ◽  
Mary C. Horne ◽  
Johannes W. Hell

Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Methods: To better determine the apparent molecular masses (MR)of the α11.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α11.2 expressed in HEK293 cells was conducted using six different region–specific antibodies against α11.2. Results: The full length form of α11.2 migrated, as expected, with an apparent MR of ~250 kDa. A shorter form of comparable prevalence with an apparent MR of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α11.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α11.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014) is at best minimal in brain tissue.


FEBS Journal ◽  
2012 ◽  
Vol 279 (13) ◽  
pp. 2357-2367 ◽  
Author(s):  
Mikhail Orel ◽  
Esteve Padrós ◽  
Joan Manyosa

1991 ◽  
Vol 46 (3-4) ◽  
pp. 189-193 ◽  
Author(s):  
Kenji Monde ◽  
Mitsuo Takasugi ◽  
Jenny A. Lewis ◽  
G. Roger Fenwick

Sliced turnip root (Brassica campestris L. ssp rapa) was irradiated for a total of 20 min with a 15 W germicidal lamp and the tissue incubated at 25 °C. The effects of such treatment on indole phytoalexins (methoxybrassinin (I); brassinin (II); cyclobrassinin (III); spirobrassinin (IV) and glucosinolates were determined using high performance liquid chromatography procedures. Accumulation of phytoalexins I - III was evident within 8 h of irradiation, whilst formation of spirobrassinin was evident only after 24 h. Maximal levels of III and IV (> 100 μg g-1 freeze dried tissue) were greater than those of I and II (27 and 17 μg g-1, respectively). The individual glucosinolate levels were affected in a complex manner; whilst most glucoinolates decreased on storage, the levels of indole glucosinolates, glucobrassicin (XI) and 1-methoxyglucobrassicin (XIII), increased until 5 to 6 days after irradiation and thereafter declined. Whilst structural features of I - IV , XI and XIII suggest close biosynthetic relationships between these classes of biologically-active indoles, further studies are needed to establish this point unambiguously.


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