Effect of Buffer Conditions and Organic Cosolvents on the Rate of Strain-Promoted Azide-Alkyne Cycloaddition.

We investigate the effect of buffer identity, ionic strength, pH, and organic cosolvents on the rate of strain-promoted azide-alkyne cycloaddition with the widely used DIBAC cyclooctyne. The rate of reaction between DIBAC and a hydrophilic azide is highly tolerant to changes in buffer conditions but is impacted by organic cosolvents. Thus, bioconjugation reactions using DIBAC can be carried out in the buffer that is most compatible with the biomolecules being labeled, but the use of organic cosolvents should be carefully considered.

Monkey adrenal chromaffin cells express α6ß4* nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) that contain α6 and ß4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6ß4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the α3ß4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express α6ß4* receptors. PCR was used to show the presence of transcripts for α6 and ß4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6ß4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and ß2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6ß4* nAChRs.

Positional scanning mutagenesis of α-conotoxin PeIA identifies critical residues that confer potency and selectivity for α6/α3ß2ß3 and α3ß2 nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) subtype α6ß2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6ß2* and α3ß2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6ß2* and α3ß2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3ß2ß3 and α3ß2 receptors. Mutagenesis of PeIA revealed that Asn(11) was located in an important position that interacts with the α6 and α3 subunits. Substitution of Asn(11) with a positively charged amino acid essentially abolished the activity of PeIA for α3ß2 but not for α6/α3ß2ß3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on α6/α3ß2ß3 than α3ß2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu(152) of the α6 subunit and Lys(152) of the α3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target α6ß2* nAChRs.

α-Conotoxin PeIA[S9H,V10A,E14N] potently and selectively blocks α6ß2ß3 versus α6ß4 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) containing α6 and ß2 subunits modulate dopamine release in the basal ganglia and are therapeutically relevant targets for treatment of neurological and psychiatric disorders including Parkinson's disease and nicotine dependence. However, the expression profile of ß2 and ß4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind α6ß2 nAChRs also potently bind the closely related α6ß4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described α-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including α6/α3ß2ß3 and α6/α3ß4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for α6/α3ß2ß3 nAChRs expressed in Xenopus oocytes (α6/α3 is a subunit chimera that contains the N-terminal ligand-binding domain of the α6 subunit). On the basis of these results, second-generation analogs were then synthesized. The final analog, PeIA[S9H,V10A,E14N], potently blocked acetylcholine-gated currents mediated by α6/α3ß2ß3 and α6/α3ß4 nAChRs with IC(50) values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to α6/α3ß2ß3 nAChRs yielded a k(off) of 0.096 ± 0.001 min(-1) and a k(on) of 0.23 ± 0.019 min(-1) M(-9). The synthesis of PeIA[S9H,V10A,E14N] demonstrates that ligands can be developed to discriminate between α6ß2 and α6ß4 nAChRs.