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Functional evidence of distinct ATP activation sites at the human P2X(7) receptor
M Klapperstück1, C Büttner, G Schmalzing
1Julius-Bernstein-Institut für Physiologie, Martin-Luther-Universität Halle-Wittenberg, Magdeburger Strasse 6, D-06097 Halle/Saale, Germany.
Abstract:
1. The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X(7) receptor (hP2X(7)), an N-terminally hexahistidyl-tagged hP2X(7) receptor (His-hP2X(7)), or a truncated His-hP2X(7) receptor (His-hP2X(7)DeltaC) 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 hP2X(7) 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 hP2X(7) 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 microM free ATP(4-), respectively. 4. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 microM. 5. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X(7) or His-hP2X(7)DeltaC. 6. Our data indicate that the hP2X(7) 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.
Insights
The human P2X(7) receptor (hP2X(7)) has two ATP activation sites with different sensitivities. N- and C-terminal domains affect low-sensitivity site activation, influencing current kinetics.
Area of Science:
- Molecular biology
- Neuroscience
- Pharmacology
Background:
- The P2X(7) receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP.
- Understanding P2X7R activation mechanisms is crucial for its role in inflammation and pain.
Purpose of the Study:
- To investigate the effect of ATP on wild-type and modified human P2X(7) receptors (hP2X(7)).
- To characterize the activation kinetics and agonist concentration dependence of hP2X(7).
Main Methods:
- Two-microelectrode voltage clamp technique in Xenopus oocytes.
- Expression of wild-type, N-terminally tagged, and C-terminally truncated hP2X(7) receptors.
Main Results:
- Wild-type hP2X(7) activation showed both exponential and linear current components.
- A biphasic dependence on ATP concentration for the exponential component suggested two high- and two low-sensitivity sites.
- The linear component showed monophasic dependence, with an apparent dissociation constant of ~200 microM ATP.
- N- and C-terminal modifications reduced or abolished low-sensitivity site contributions.
Conclusions:
- The hP2X(7) receptor possesses at least two distinct ATP activation sites with a 50-fold difference in sensitivity.
- Site occupation influences activation and deactivation kinetics.
- Both N- and C-terminal domains are critical for low-sensitivity ATP activation but not high-sensitivity activation.