Earlier, a family of G protein-coupled receptors, termed T2Rs, was identified in the rodent and human genomes through data mining. It was suggested that these receptors mediate bitter taste perception. Analysis of the human genome revealed that the hT2R family is composed of 25 members. However, bitter ligands have been identified for only three human receptors so far. Here we report identification of two novel ligand-receptor pairs. hT2R61 is activated by 6-nitrosaccharin, a bitter derivative of saccharin. hT2R44 is activated by denatonium and 6-nitrosaccharin. Activation profiles for these receptors correlate with psychophysical data determined for the bitter compounds in human studies. Functional analysis of hT2R chimeras allowed us to identify residues in extracellular loops critical for receptor activation by ligands. The discovery of two novel bitter ligand-receptor pairs provides additional support for the hypothesis that hT2Rs mediate a bitter taste response in humans.

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of ␣, ␤, and ␥ subunits, belongs to the ENaC/ degenerin family of ion channels and forms the principal route for apical Na ؉ entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the ␤ subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of ␣␤G37S␥ hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na ؉ flux across epithelial membranes is clinically desirable.

The series of phenoxide complexes cis-L 4 Os(H)OC 6 H 4 Z (L ) PMe 3 ; Z ) H, OMe, CF 3 , NH 2 , CN), anilides cis-L 4 Os(H)NHC 6 H 4 Z (Z ) H, OMe, CF 3 ), and thiophenoxides cis-L 4 Os(H)-SC 6 H 4 Z (Z ) H, OMe) have been prepared by treatment of fac-L 3 Os(H)(η 2 -CH 2 PMe 2 ) with the corresponding neutral arene. 1 H NMR spectra of coordinated phenoxides and thiophenoxides show rapid phenyl group rotation, while that of the anilides is slow. Rates and stereochemistry of substitution of P(CH 3 ) 3 (L) by P(CD 3 ) 3 (L′) were determined by 31 P NMR in benzene at 80°C. Anilides substitute first only in the mutually trans sites (a sites) with cleanly first-order kinetics and subsequently into the site trans to the anilide (site c). The latter shows non-first-order behavior that is accurately modeled by iterative kinetics calculations using a mechanism of L dissociation only from the a sites presumably to give a quasi-trigonal-bipyramidal intermediate stabilized by π-electron donation from the anilide lone pair. A three-point Hammett plot against σ p yielding F ) -1.8 is consistent with transition state stabilization by π-donation. Association of L′ occurs only into site a, but subsequent substitution allows the resident L′ to move into site c. Thiophenoxides exhibit substitution rates and stereochemical patterns very similar to those of the anilides and are believed to proceed by the same mechanism. Phenoxide complexes incorporate L′ into all sites, with each site incorporating phosphine independently of the others since exchange rates at all sites are first order. A Hammett plot of exchange rates against sigma minus (σ -) is somewhat scattered (R 2 ) 0.96) but exhibits a positive slope F -) +0.36. Phenoxide dissociation is postulated, but the fact that substantial concentrations of intermediates partially substituted in all positions is seen during the reaction is inconsistent with ratedetermining phenoxide dissociation. An ionization preequilibrium followed by slower phosphine exchange steps in the ion pair is postulated. Treatment of L 4 Os(H)(OC 6 H 4 CN) with excess L in propylene carbonate, DMSO-d 6 , DMF, or CD 3 NO 2 at 80°C all resulted in conversions to [L 5 OsH][OAr]. These results suggest that low steady-state concentrations of [L 5 OsH][OAr] ion pairs in benzene are possible, consistent with an ion pair mechanism for ligand exchange.

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