Background: Nicotinic acetylcholine receptors (nAChRs) are involved in nicotine addiction and some neurological disorders. Results: A novel positive allosteric modulator potentiates activation through the C-tail of one ␣4 subunit but requires two ␣4 to reactivate desensitized nAChRs. Conclusion: Higher occupancy in allosteric sites promotes nAChR opening and alleviates desensitization. Significance: These ␣4 modulators may be useful for basic and clinical applications.

Sodium influx through voltage-gated sodium channels (VGSCs) coupled with balanced removal of sodium ions via Na+, K+-ATPase is a major determinant of cellular homeostasis and intracellular ionic concentration. Interestingly, many metastatic carcinomas express high levels of these channels. We hypothesized that if excess VGSCs are activated and Na+, K+-ATPase is simultaneously blocked, the intracellular Na+ concentration should increase, resulting in water movement into the cell, causing swelling and lytic cell death. MDA-MB-231 breast cancer cells over-express VGSCs by 7-fold. To test our hypothesis, we treated these cells in vitro with the Na+, K+-ATPase blocker, ouabain, and then stimulated with a sublethal electric current. For in vivo histologic and survival studies, MDA-MB-231 xenografts were established in Nu/J mice. Mice injected with saline or ouabain were electrically stimulated with trains of 10 msec 10V DC pulses. Within seconds to minutes, the cells swelled and lysed. MCF-10a cells, which express normal VGSCs levels, were unaffected by this treatment. Cells from the weakly-malignant cell line, MCF-7, which express 3-fold greater VGSCs than MCF-10a cells, displayed an intermediate time-to-lysis. The rate of lysis correlated directly with the degree of sodium channel expression and malignancy. We also demonstrated efficacy in cell lines from prostate, colon and lung carcinomas. Treated MDA-MB-231 xenografts showed 60–80% cell death. In survival studies, TOL-treated mice showed significantly slower tumor growth vs. controls. These results are evidence that this ”targeted osmotic lysis” represents a novel method for selectively killing cancer cells and warrants further investigation as a potential treatment for advanced and end-stage breast cancer.

Edited by Mike Shipston Nicotinic acetylcholine receptor (nAChR) ligands that lack agonist activity but enhance activation in the presence of an agonist are called positive allosteric modulators (PAMs). nAChR PAMs have therapeutic potential for the treatment of nicotine addiction and several neuropsychiatric disorders. PAMs need to be selectively targeted toward certain nAChR subtypes to tap this potential. We previously discovered a novel PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which selectively potentiates the opening of ␣4␤2*, ␣2␤2*, ␣2␤4*, and (␣4␤4) 2 ␣4 nAChRs and reactivates some of these subtypes when desensitized (* indicates the presence of other subunits). We located the Br-PBTC-binding site through mutagenesis and docking in ␣4. The amino acids Glu-282 and Phe-286 near the extracellular domain on the third transmembrane helix were found to be crucial for Br-PBTC's PAM effect. E282Q abolishes Br-PBTC potentiation. Using (␣4 E282Q ␤2) 2 ␣5 nAChRs, we discovered that the trifluoromethylated derivatives of Br-PBTC can potentiate channel opening of ␣5-containing nAChRs. Mutating Tyr-430 in the ␣5 M4 domain changed ␣5-selectivity among Br-PBTC derivatives. There are two kinds of ␣4 subunits in ␣4␤2 nAChRs. Primary ␣4 forms an agonist-binding site with another ␤2 subunit. Accessory ␣4 forms an agonist-binding site with another ␣4 subunit. The pharmacological effect of Br-PBTC depends both on its own and agonists' occupancy of primary and accessory ␣4 subunits. Br-PBTC reactivates desensitized (␣4␤2) 2 ␣4 nAChRs. Its full efficacy requires intact Br-PBTC sites in at least one accessory and one primary ␣4 subunit. PAM potency increases with higher occupancy of the agonist sites. Br-PBTC and its derivatives should prove useful as ␣ subunit-selective nAChR PAMs.