Ectodomain lysines and suramin block of P2X1 receptors

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.

FIGURE 1.

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.

FIGURE 2.

Introduction of lysine residues into the mouse receptor can increase sensitivity to suramin. A, representative traces of membrane currents evoked by ATP (10 μm), applied for 1 s as indicated by the horizontal bars. Left panels, control. Right panels, in suramin (10 μm). B, ATP concentration-response curves for wild type mouse P2X₁ receptors and for receptors with one or four lysines introduced at the positions indicated. Open symbols, control. Solid symbols, in presence of suramin (10 μm). Note the increased effectiveness of suramin as an antagonist in the case of mouse P2X₁[E138K] and of mouse P2X₁[4K] receptors.

FIGURE 3.

Introduction of lysine residues into the mouse receptor can increase sensitivity to NF449. A, representative traces of membrane currents evoked by ATP (10 μm), applied for 1 s as indicated by the horizontal bars. Left panels, control. Center and right panels, in NF449 (3 and 300 nm). B, ATP concentration-response curves for wild type mouse P2X₁ receptors and for receptors with one or four lysines introduced at the positions indicated. Open symbols, control. Solid symbols, in presence of NF449 (3 and 300 nm). Note the increased effectiveness of NF449 (3 nm) as an antagonist in the case of mouse P2X₁[E138K] and of mouse P2X₁[4K] receptors.

FIGURE 4.

Removal of lysine residues from the human mouse receptor can reduce sensitivity to suramin. A, representative traces of membrane currents evoked by ATP (10 μm), applied for 1 s as indicated by the horizontal bar. Left panels, control. Right panels, in suramin (10 μm). B, ATP concentration-response curves for wild type mouse P2X₁ receptors and for receptors with one or four lysines removed at the positions indicated. Open symbols, control. Solid symbols, in presence of suramin (10 μm). Note the decreased effectiveness of suramin as an antagonist in the case of human P2X₁[K138E] and also for human P2X₁[No K] receptors.

FIGURE 5.

Removal of lysine residues from the human can reduce sensitivity to NF449. A, representative traces of membrane currents evoked by ATP (10 μm), applied for 1 s as indicated by the horizontal bar. Left panels, control. Center and right panels, in NF449 (3 and 300 nm). B, ATP concentration-response curves for wild type human P2X₁ receptors and for receptors with one or four lysines removed at the positions indicated. Open symbols, control. Solid symbols, in presence of NF449 (3 and 300 nm). Note the decreased effectiveness of NF449 (300 nm) as an antagonist in the case of human P2X₁[K138E] and also for human P2X₁[No K] receptors.