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 es timated by power saturating thp EPR spectrum nf a membrane bound N-14 spin-labeled amphiphile in the presence of a neutral or positively cha rged N-15 labeled aqueous spin probe. The potentials that are measured are in good agreement with other probe measurements and with the pred ictions of the Gouy-Chapman-Stern theory, indicating that this is a va lid approach to determine electrostatic potentials. A spin-labeled aff inity probe based on maleimidobenzyltrimethylammonium was synthesized and could be derivatized to a sulfhydryl near either agonist site on t he nicotinic acetylcholine receptor. The amplitudes of motion of the s pin-probe on the ns time scale are significantly different when the tw o 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 poten tial of -15 mV. Two other sulfhydryl-specific probes were used to labe l this site in reconstituted receptor containing membranes. These prob es show less contact with the receptor and reduced electrostatic poten tials, indicating that there is a strong spatial dependence to the pot ential at the agonist site. This work demonstrates that power saturati on EPR provides a general method that can be used to estimate electros tatic potentials at any specifically spin-labeled macromolecular site. (C) 1997 Elsevier Science B.V.