Computed Pore Potentials of the Nicotinic Acetylcholine Receptor

Abstract

Electrostatic surface potentials in the vestibule of the nicotinic acetylcholine receptor (nAChR) were computed from structural models using the University of Houston Brownian Dynamics program to determine their effect on ion conduction and ionic selectivity. To further determine whether computed potentials accurately reflect the electrostatic environment of the channel, the potentials were used to predict the rate constants for diffusion-enhanced fluorescence energy transfer; the calculated energy transfer rates are directly comparable with those determined experimentally (see companion article by Meltzer et al. in this issue). To include any effects on the local potentials by the bound acceptor fluorophore crystal violet, its binding site was first localized within the pore by fluorescence energy transfer measurements from dansyl-C6-choline bound to the agonist sites and also by simulations of binding using Autodock. To compare the computed potentials with those determined experimentally, we used the predicted energy transfer rates from Tb3+ chelates of varying charge to calculate an expected potential using the Boltzmann relationship. This expected potential (from -20 to -40 mV) overestimates the values determined experimentally (from -10 to -25 mV) by two- to fourfold at similar conditions of ionic strength. Although the results indicate a basic discrepancy between experimental and computed surface potentials, both methods demonstrate that the vestibular potential has a relatively small effect on conduction and selectivity.

Rings of Charge within the Extracellular Vestibule Influence Ion Permeation of the 5-HT3A Receptor

“…Such stabilization indicates the presence of an energy well that would be anticipated to affect both inward and outward ionic movements. Energy wells within the extracellular vestibule are also apparent as negative vestibular pore potentials computed for the Torpedo nACh receptor ( 23 , 24 ). We speculate that the decrease or possibly reversal in pore potential caused by the D127N and D127K mutations, respectively, removes a force that facilitates both inwardly and outwardly directed ion fluxes or introduces a barrier against such movements.…”

“…see Refs. 23 and 24 ). By contrast, a homology model of the GABA A receptor indicates the ECD to concentrate anions in preference to cations ( 25 ).…”

FRET in membrane biophysics: an overview

“…This is the reason why rapid-diffusion limit FRET found application in the measurement of the distance of closest D–A approach (where typically the A species is a protein), in the 1980s (see Table 1 for examples). Additionally, FRET-enhanced diffusion of charged D/A species is sensitive to the electrostatic potential, and therefore can be used to determine membrane potentials (Meltzer et al, 2006). Surprisingly, recent applications in membranes have been very few, which led to the spirited comment “Since the mid 1980s, the technique [diffusion-enhanced FRET] seems to have remained dormant waiting for the right opportunity to spring back to life” (Fairclough, 2006).…”

Structural answers and persistent questions about how nicotinic receptors work

“…M4 appeared to link the lipid environment to nAChR gating (374). Electrostatic forces facilitating ion flow through the pore likely arise from the transmembrane domain and to a much lesser extent from longer range interactions from the extracellular domain or large cytoplasmic loop (375,376).…”