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Functional evidence of distinct ATP activation sites at the human P2X₇ receptor

Manuela Klapperstück, Cora Büttner *, Günther Schmalzing *† and Fritz Markwardt

1. The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X₇ receptor (hP2X₇), an N-terminally hexahistidyl-tagged hP2X₇ receptor (His-hP2X₇), or a truncated His-hP2X₇ receptor (His-hP2X₇C) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique.
2. The activation time course of the wild-type hP2X₇ receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component.
3. The amplitude of the exponentially activating current component of the wild-type hP2X₇ receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity non-cooperative activation sites with apparent dissociation constants of about 4 and 200 µM free ATP^4-, respectively.
4. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 µM.
5. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X₇ or His-hP2X₇C.
6. Our data indicate that the hP2X₇ receptor possesses at least two types of activation sites, which differ in ATP^4- sensitivity by a factor of 50. The degree of occupation of these two sites influences both activation and deactivation kinetics. Both N- and C-terminal domains appear to be important determinants of the current elicited by activation of the sites with low ATP sensitivity, but not for that mediated by the highly ATP-sensitive sites.

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