Sim JA, Broomhead HE, North RA
Ectodomain lysines and suramin block of P2X1 receptors. [JOURNAL ARTICLE]
J Biol Chem 2008 Sep 2.
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 receptors P2X(1) receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP recorded. ATP (10 nM - 100 muM) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X1 receptors. Suramin (10 muM) and NF449 (3 - 300 nM) effectively blocked the human receptor but had little effect at 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), 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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