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Ectodomain lysines and suramin block of P2X1 receptors.

Sim JA, Broomhead HE, North RA - J. Biol. Chem. (2008)

Bottom Line: ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors.The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449.The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom. joan.sim@manchester.ac.uk

ABSTRACT
P2X(1) receptors belong to a family of cation channels gated by extracellular ATP; they are found inter alia in smooth muscle, platelets, and immune cells. Suramin has been widely used as an antagonist at P2X receptors, and its analog 4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))] tetrakis-benzene-1,3-disulfonic acid (NF449) is selective for the P2X(1) subtype. Human and mouse P2X(1) receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors. Suramin (10 microm) and NF449 (3-300 nM) effectively blocked the human receptor. Suramin had little effect on the mouse receptor. Suramin and NF449 are polysulfonates, with six and eight negative charges, respectively. We hypothesized that species differences might result from differences in positive residues presented by the large receptor ectodomain. Four lysines in the human sequence (Lys(111), Lys(127), Lys(138), and Lys(148)) were changed individually and together to their counterparts in the mouse sequence. The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449. Conversely, when lysine was introduced into the mouse receptor, the sensitivity to block by suramin and NF449 was much increased for E138K, but not for Q111K, Q127K, or N148K. The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

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Four lysines in the human P2X1 receptor are missing in the mouse ortholog. Comparison of sequences of the human and mouse receptors in the ectodomain segment from residues 111 to 148 (numbering is the same in both species) shows four positions where lysine is replaced by uncharged polar or negative amino acid.
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fig1: Four lysines in the human P2X1 receptor are missing in the mouse ortholog. Comparison of sequences of the human and mouse receptors in the ectodomain segment from residues 111 to 148 (numbering is the same in both species) shows four positions where lysine is replaced by uncharged polar or negative amino acid.

Mentions: In the amino acid sequences of the human and mouse P2X1 receptors (SwissProt: mouse P51576 and human P51575), there are 40 differences in 399 residues, 33 of which are in the ectodomain. In four cases, there are lysine residues in the human sequence that correspond in position to neutral or negatively charged residues in the mouse sequence. These are clustered in a part of the protein ectodomain that begins some 60 amino acids after the end of the first transmembrane domain (positions 111, 127, 138, and 148; numbering is the same for mouse and human) (Fig. 1). Suramin and NF449 bear fixed negative charges by virtue of their polysulfonates (six in suramin and eight in NF449). We therefore hypothesized that the difference in suramin sensitivity between the two species resulted from the different presentation of positively charged residues by the two receptors and elected to test this in the first instance by focusing on the region Lys111 to Lys148. We tested this hypothesis by systematically substituting each of the four lysines in the human sequence by its counterpart in the mouse and vice versa.


Ectodomain lysines and suramin block of P2X1 receptors.

Sim JA, Broomhead HE, North RA - J. Biol. Chem. (2008)

Four lysines in the human P2X1 receptor are missing in the mouse ortholog. Comparison of sequences of the human and mouse receptors in the ectodomain segment from residues 111 to 148 (numbering is the same in both species) shows four positions where lysine is replaced by uncharged polar or negative amino acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2573084&req=5

fig1: Four lysines in the human P2X1 receptor are missing in the mouse ortholog. Comparison of sequences of the human and mouse receptors in the ectodomain segment from residues 111 to 148 (numbering is the same in both species) shows four positions where lysine is replaced by uncharged polar or negative amino acid.
Mentions: In the amino acid sequences of the human and mouse P2X1 receptors (SwissProt: mouse P51576 and human P51575), there are 40 differences in 399 residues, 33 of which are in the ectodomain. In four cases, there are lysine residues in the human sequence that correspond in position to neutral or negatively charged residues in the mouse sequence. These are clustered in a part of the protein ectodomain that begins some 60 amino acids after the end of the first transmembrane domain (positions 111, 127, 138, and 148; numbering is the same for mouse and human) (Fig. 1). Suramin and NF449 bear fixed negative charges by virtue of their polysulfonates (six in suramin and eight in NF449). We therefore hypothesized that the difference in suramin sensitivity between the two species resulted from the different presentation of positively charged residues by the two receptors and elected to test this in the first instance by focusing on the region Lys111 to Lys148. We tested this hypothesis by systematically substituting each of the four lysines in the human sequence by its counterpart in the mouse and vice versa.

Bottom Line: ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors.The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449.The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom. joan.sim@manchester.ac.uk

ABSTRACT
P2X(1) receptors belong to a family of cation channels gated by extracellular ATP; they are found inter alia in smooth muscle, platelets, and immune cells. Suramin has been widely used as an antagonist at P2X receptors, and its analog 4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))] tetrakis-benzene-1,3-disulfonic acid (NF449) is selective for the P2X(1) subtype. Human and mouse P2X(1) receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors. Suramin (10 microm) and NF449 (3-300 nM) effectively blocked the human receptor. Suramin had little effect on the mouse receptor. Suramin and NF449 are polysulfonates, with six and eight negative charges, respectively. We hypothesized that species differences might result from differences in positive residues presented by the large receptor ectodomain. Four lysines in the human sequence (Lys(111), Lys(127), Lys(138), and Lys(148)) were changed individually and together to their counterparts in the mouse sequence. The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449. Conversely, when lysine was introduced into the mouse receptor, the sensitivity to block by suramin and NF449 was much increased for E138K, but not for Q111K, Q127K, or N148K. The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

Show MeSH
Related in: MedlinePlus