State-Dependent Cross Inhibition

State-dependent cross inhibition

between transmitter-gated cation channels

*Baljit S. Khakh, Xiaoping Zhou, *Jason Sydes, James J. Galligan & *Henry A. Lester

*Division of Biology, California Institute of Technology, Pasadena.

Department of Pharmacology and Toxicology and the Neuroscience Program, Michigan State University, East Lansing.

Manuscript LO2152A LA/sad

Supplementary Information

Supplementary Table 1. ATP and ACh potencies from cells expressing various combinations of wild type and mutant 34 nicotinic and P2X2 channels.

EC50 (M) / nH / n
IATP / 34 + P2X2 / 3.7  0.3 / 2.3  0.2 / 6
3 + P2X2 / 3.1  0.1 / 1.8  0.03 / 3
4 + P2X2 / 2.9  0.08 / 1.8  0.03 / 4
P2X2 / 3.8  0.8 / 1.6  0.06 / 4
3Ser9’Leu)4 + P2X2 / 8.1  0.9 / _ / 4
IACh / 34 / 20.1  2. / 1.4  0.07 / 4
34 + P2X2 / 21.4  1.9 / 1.0  0.03 / 4
3Ser9’Leu)4 / 0.1  0.001 / _ / 5
3Ser9’Leu4) + P2X2 / 0.1  0.03 / _ / 4

- Indicates that a reliable Hill slope could not be determined for recordings with the Ser9’Leu mutant channels because of poor regressions.

Supplementary Figure 1. Properties of channels formed by co-expression of P2X2 and 34 nicotinic channels.

(a) Concentration-effect curves for ATP at P2X2 channels, either alone or expressed with 3, 4 or 34 channels. (b) Concentration-effect curves for ACh from oocytes injected with cRNA for34 channels, 3, 4 or 34 and P2X2 channels. (c) Concentration-effect curves for ACh from oocytes injected with cRNA for 34 and P2X2 channels. ACh concentration-effect curves were determined either alone and in the presence of 10 M ATP. (d) Normalized ACh concentration-effect curves with and without 10 M ATP from experiments illustrated in panel c.

Supplementary Figure 2. ATP does not gate or modulate 34 channels, and ACh does not gate or modulate P2X2 channels.

(a) Current-voltage relations during application of ATP, ACh and ATP/ACh in cells expressing 34 channels alone (n = 4). (b) Current-voltage relations during application of ATP, ACh and ATP/ACh in cells expressing P2X2 channels alone (n=3).

Supplementary Figure 3. Coexpression of 34 channels affects P2X2 ion selectivity dynamics.

(a) IATP current-voltage relations determined every 300 ms from a P2X2 channel expressing cell with NMDG+ as the extracellular cation. (b) IATP current-voltage relations determined every 300 ms from a P2X2 and 34 channel expressing cell with NMDG+ as the extracellular cation. (c) Time-dependence of the shift in NMDG+ reversal potential for IATP for 10 cells expressing P2X2 channels alone. (d) Time-dependence of the shift in NMDG+ reversal potential for IATP for 10 cells expressing P2X2 channels and 34 channels. (e) Amplitude of the initial outward current peak in NMDG+ at –60 mV, termed I1, for cells expressing just P2X2 channels and those that co-express P2X2 and 34 channels. (f) Amplitude of the final inward current peak in NMDG+, termed I2, for cells expressing just P2X2 channels and those that co-express P2X2 and 34 channels.

Supplementary Figure 4. Additivity between P2X2 mediated currents and other receptor mediated currents. (a) Trace from a cell expressing P2X2, m2 muscarinic receptors and GIRK1/4 channels. 100 M ATP evoked a P2X2 mediated current that was 100% additive with the ACh current in this, and all other cells (n = 9; panel b). (c) Rectification properties of P2X2 channels. (d) Summary of data from cells co-expressing P2X2 channels and Kv1.3 channels in oocytes (n = 9). Kv channels were activated by steps (from –60 mV to + 40 mV) and the amplitude of the steady state current is shown in the upper panel. The lower panel shows the current at –60 mV before, during, and after 100 M ATP application. (e) Representative traces of Kv1.3 currents before and after ATP (superimposed in left hand panel), and during ATP application (right hand panel).