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J. Biol. Chem., Vol. 283, Issue 44, 29841-29846, October 31, 2008

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Ectodomain Lysines and Suramin Block of P2X₁ Receptors^*

From the Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom

P2X₁ 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 P2X₁ subtype. Human and mouse P2X₁ receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nM to 100 µM) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X₁ receptors. Suramin (10 µM) 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 (Lys¹¹¹, Lys¹²⁷, Lys¹³⁸, and Lys¹⁴⁸) 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 P2X₁ receptors.

Received for publication, April 1, 2008 , and in revised form, August 11, 2008.

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^* This work was supported by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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¹ To whom correspondence should be addressed: Faculty of Life Sciences, University of Manchester, Michael Smith Bldg., Oxford Road, Manchester M13 9PT, UK. Fax: 44-161-275-1497; E-mail: joan.sim{at}manchester.ac.uk.

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