Permeant but not impermeant divalent cations enhance activation of nondesensitizing α₇ nicotinic receptors

Abstract

Neuronal alpha(7) nicotinic acetylcholine receptors (nAChRs) are permeable to Ca(2+) and other divalent cations. We characterized the modulation of the pharmacological properties of nondesensitizing mutant (L(247)T and S(240)T/L(247)T) alpha(7) nAChRs by permeant (Ca(2+), Ba(2+), and Sr(2+)) and impermeant (Cd(2+) and Zn(2+)) divalent cations. alpha(7) receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh, whereas impermeant divalent cations decreased potency and maximal efficacy. The antagonist dihydro-beta-erythroidine (DHbetaE) was a strong partial agonist of L(247)T and S(240)T/L(247)T alpha(7) receptors in the presence of divalent cations but was a weak partial agonist in the presence of impermeant divalent cations. Mutation of the "intermediate ring" glutamates (E(237)A) in L(247)T alpha(7) nAChRs eliminated Ca(2+) conductance but did not alter the Ca(2+)-dependent increase in ACh potency, suggesting that site(s) required for modulation are on the extracellular side of the intermediate ring. The difference between permeant and impermeant divalent cations suggests that sites within the pore are important for modulation by divalent cations.

Role of the Outer β-Sheet in Divalent Cation Modulation of α7 Nicotinic Receptors

“…Oocytes were surgically removed and prepared from female X. laevis in accordance with the University of North Carolina Institutional Animal Care and Use Committee guidelines as described previously (Eddins et al, 2002a). cRNA was prepared using the T7 RNA polymerase and mMessage mMachine kit (Ambion, Austin, TX) as described by the manufacturer.…”

“…Currents were low-pass-filtered at 50 or 125 Hz and were sampled at 250 Hz. Agonist dose-response curves were obtained as described previously (Eddins et al, 2002a), and data were fit to the Hill equation using Origin software (OriginLab Corp., Northampton, MA).…”

“…Previous studies showed that mutation of the conserved glutamate at 172 eliminated divalent modulation of ␣7 AChRs, demonstrating that this residue is a critical component of a divalent allosteric binding site (Galzi et al, 1996;Eddins et al, 2002a). To test whether this site was involved in the activation by divalent cations of K 144 C/T 198 C ␣7 AChR, we constructed ␣7 receptors with Glu 172 mutated to glutamine in addition to K 144 C and T 198 C mutations.…”

Agonist-Induced Conformational Changes in the Extracellular Domain of α7 Nicotinic Acetylcholine Receptors

“…Oocytes were surgically removed from female X. laevis frogs in accordance with University of North Carolina Institutional Animal Care and Use Committee guidelines. Oocytes were treated with collagenase to remove follicular cells and were maintained using standard procedures as described previously (Eddins et al, 2002a). cRNA was prepared using the T7 RNA polymerase and mMessage mMachine kit (Ambion, Austin, TX) as described by the manufacturer.…”

“…Agonist dose-response curves were obtained as described previously (Eddins et al, 2002a), and data were fit to the Hill equation using Prism software (GraphPad Software Inc., San Diego, CA). Inhibitory dose-response curves were obtained in the presence of approximate EC 50 concentrations of ACh.…”