Estimating the electrostatic potential at the acetylcholine receptor agonist site using power saturation EPR

Continuous wave EPR power saturation was used to measure electrostatic potentials at spin-labeled sites. Membrane surface potentials were estimated by power saturating the EPR spectrum of a membrane bound 14N spin-labeled amphiphile in the presence of a neutral or positively charged 15N labeled aqueous spin probe. The potentials that are measured are in good agreement with other probe measurements and with the predictions of the Gouy-Chapman-Stern theory, indicating that this is a valid approach to determine electrostatic potentials. A spin-labeled affinity probe based on maleimidobenzyltrimethylammonium was synthesized and could be derivatized to a sulfhydryl near either agonist site on the nicotinic acetylcholine receptor. The amplitudes of motion of the spin-probe on the ns time scale are significantly different when the two labeled sites are compared, and the probe is more restricted in its motion when attached to the more easily labeled site. When attached to this agonist site, power saturation EPR yields an electrostatic potential of -15 mV. Two other sulfhydryl-specific probes were used to label this site in reconstituted receptor containing membranes. These probes show less contact with the receptor and reduced electrostatic potentials, indicating that there is a strong spatial dependence to the potential at the agonist site. This work demonstrates that power saturation EPR provides a general method that can be used to estimate electrostatic potentials at any specifically spin-labeled macromolecular site.