The molecular mechanisms that couple agonist binding to the gating of Cys-loop ionotropic receptors are not well understood. The crystal structure of the acetylcholine (ACh) binding protein has provided insights into the structure of the extracellular domain of nicotinic receptors and a framework for testing mechanisms of activation. Key ligand binding residues are located at the C-terminal end of the ␤9 strand. At the N-terminal end of this strand (loop 9) is a conserved glutamate [E 172 in chick ␣7 nicotinic acetylcholine receptors (nAChRs)] that is important for modulating activation. We hypothesize that agonist binding induces the movement of loop 9. To test this, we used the substituted-cysteine accessibility method to examine agonist-dependent changes in the modification of cysteines introduced in loop 9 of L 247 T ␣7 nAChRs. In the absence of agonist, ACh-evoked responses of E 172 C/L 247 T ␣7 nAChRs were inhibited by 2-trimethylammonioethylmethane thiosulfonate (MTSET). Agonist coapplication with MTSET reduced the extent and rate of modification. The dose-dependence of ACh activation was nearly identical with that of ACh-dependent protection from modification. ACh increased the inhibition by methanethiosulfonate reagents of N 170 C and did not change inhibition of G 171 C receptors. The antagonist dihydro-␤-erythroidine did not mimic the effects of ACh. Combined with a structural model, the data suggest that receptor activation includes subunit rotation and/or intrasubunit conformational changes that move N 170 to a more accessible position and E 172 to a more protected position away from the vestibule. Thus, loop 9, located near the junction between the extracellular and transmembrane domains, participates in conformational changes triggered by ligand binding.

␣-7 Nicotinic acetylcholine receptors (AChRs) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick ␣7 AChR, this modulation involves a conserved glutamate in loop 9 (Glu 172 ) that undergoes agonistdependent movements during activation. From these observations, we hypothesized that movements of the nearby ␤-sheet formed by the ␤7, ␤9, and ␤10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis, we examined functional properties of cysteine mutations of the ␤7 and ␤10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the ␤7/

N-methyl-D-aspartate receptors (NMDARs) are Ca(2+)-permeable, ligand-gated, nonselective cation channels that function as neuronal synaptic receptors but which are also expressed in multiple peripheral tissues. Here, we show for the first time that NMDAR subunits NR3a and NR3b are highly expressed in the neonatal kidney and that there is continued expression of NR3a in the renal medulla and papilla of the adult mouse. NR3a was also expressed in mIMCD-3 cells, where it was found that hypoxia and hypertonicity upregulated NR3a expression. Using short-hairpin (sh) RNA-based knockdown, a stable inner medullary collecting duct (IMCD) cell line was established that had ∼80% decrease in NR3a. Knockdown cells exhibited an increased basal intracellular calcium concentration, reduced cell proliferation, and increased cell death. In addition, NR3a knockdown cells exhibited reduced water transport in response to the addition of vasopressin, suggesting an alteration in aquaporin-2 (AQP2) expression/function. Consistent with this notion, we demonstrate decreased surface expression of glycosylated AQP2 in IMCD cells transfected with NR3a shRNA. To determine whether this also occurred in vivo, we compared AQP2 levels in wild-type vs. in NR3a(-/-) mice. Total AQP2 protein levels in the outer and inner medulla were significantly reduced in knockout mice compared with control mice. Finally, NR3a(-/-) mice showed a significant delay in their ability to increase urine osmolality during water restriction. Thus NR3a may play a renoprotective role in collecting duct cells. Therefore, under conditions that are associated with high vasopressin levels, NR3a, by maintaining low intracellular calcium levels, protects the function of the principal cells to reabsorb water and thereby increase medullary osmolality.

. These permeant divalent cations interact with slowly desensitizing L 247 T ␣7 nAChRs to increase the potency and maximal efficacy of ACh, increase the efficacy of dihydro-␤-erythroidine (DH␤E), and increase agonistindependent activity. Mutation of glutamate 172 (E 172 ) 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 E 172 C/L 247 T ␣7 nAChRs by Ͼ90%, demonstrating that E 172 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 E 172 projects toward the lumen of the extracellular vestibule. The observations that E 172 was essential for divalent cation modulation of L 247 T ␣7 nAChRs and was accessible to permeating ions suggest that this residue participates in coupling ion permeation with modulation of receptor activity.

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