The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current–voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of ∼30 pS at −100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was ∼150 mM at 0 mV and voltage dependent. The binding site appeared to be ∼0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 μM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of ∼7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose–response curve.
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1 May 1999
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May 01 1999
Single Channel Properties of P2X2 Purinoceptors
Shinghua Ding,
Shinghua Ding
From the *Department of Chemical Engineering and ‡Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214
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Frederick Sachs
Frederick Sachs
From the *Department of Chemical Engineering and ‡Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214
Search for other works by this author on:
Shinghua Ding
From the *Department of Chemical Engineering and ‡Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214
Frederick Sachs
From the *Department of Chemical Engineering and ‡Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214
Received:
December 10 1998
Accepted:
March 09 1999
Online ISSN: 1540-7748
Print ISSN: 0022-1295
1999
J Gen Physiol (1999) 113 (5): 695–720.
Article history
Received:
December 10 1998
Accepted:
March 09 1999
Citation
Shinghua Ding, Frederick Sachs; Single Channel Properties of P2X2 Purinoceptors . J Gen Physiol 1 May 1999; 113 (5): 695–720. doi: https://doi.org/10.1085/jgp.113.5.695
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