Neuronal ₇ nicotinic ACh receptors (nAChRs) are permeable to and modulated by Ca²⁺, Ba²⁺, and Sr²⁺. These permeant divalent cations interact with slowly desensitizing L²⁴⁷T ₇ nAChRs to increase the potency and maximal efficacy of ACh, increase the efficacy of dihydro--erythroidine (DHE), and increase agonist-independent activity. Mutation of glutamate 172 (E¹⁷²) to glutamine or cysteine eliminated these effects of permeant divalent cations. 2-(Trimethylammonium)ethyl methanethiosulfonate (MTSET), a cysteine-modifying reagent directed at water-accessible thiols, inhibited ACh-evoked currents of E¹⁷²C/L²⁴⁷T ₇ nAChRs by >90%, demonstrating that E¹⁷² was accessible to permeant ions. The data are consistent with a model of ₇ receptors, derived from the crystal structure of the ACh binding protein (AChBP) from Lymnaea stagnalis, in which E¹⁷² projects toward the lumen of the extracellular vestibule. The observations that E¹⁷² was essential for divalent cation modulation of L²⁴⁷T ₇ nAChRs and was accessible to permeating ions suggest that this residue participates in coupling ion permeation with modulation of receptor activity.