The SLC26 gene family encodes multifunctional transport proteins in numerous tissues and organs. Some paralogs function as anion exchangers, others as anion channels, and one, prestin (SLC26A5), represents a membrane-bound motor protein in outer hair cells of the inner ear. At present, little is known about the molecular basis of this functional diversity. We studied the subunit stoichiometry of one bacterial, one teleost, and two mammalian SLC26 isoforms expressed in Xenopus laevis oocytes or in mammalian cells using blue native PAGE and chemical cross-linking. All tested SLC26s are assembled as dimers composed of two identical subunits.Co-expressionoftwomutantprestinswithdistinctvoltagedependent capacitances results in motor proteins with novel electrical properties, indicating that the two subunits do not function independently. Our results indicate that an evolutionarily conserved dimeric quaternary structure represents the native and functional state of SLC26 transporters.

Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6R␣ and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFP-IL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6R␣, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6⅐IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.Cytokines are important mediators in the regulation of immune responses and inflammation. Dysregulated cytokine signaling leads to chronic inflammation and cancer. Therefore, pro-inflammatory cytokines, such as tumor necrosis factor (TNF) 2 and interleukin-1 and -6, have been identified as promising therapeutic targets. First approaches to specifically block the action of pro-inflammatory cytokines have focused on the use of neutralizing antibodies against a specific cytokine or its receptor. Only recently, the value of soluble cytokine receptors as cytokine antagonists for the treatment of inflammatory diseases has been fully recognized. Pro-inflammatory cytokines signal through receptor proteins consisting of an extracellular part, a single transmembrane region and a cytoplasmic domain. Ligand binding to the extracellular part of the receptor results in the activation of signal transduction cascades by the cytoplasmic domain. Soluble receptors consisting only of the extracellular part are potent inhibitors of cytokine activity. They bind the cytokine with the same specificity and affinity as the membranebound receptors without eliciting an intracellular signal. A dimeric form of the soluble TNF receptor is currently used for the treatment of inflammatory diseases caused by elevated TNF expression (1).Most cytokines signal through heteromeric receptor complexes consisting of two or more different r...

The FDA-approved drug bupropion has been prescribed as an antidepressant (Wellbutrin) for over three decades, and more recently as a smoking cessation aid (Zyban). The presumed mechanism of action of bupropion was inhibition of norephinephrine and dopamine reuptake by their respective transporters. Recently, bupropion's non-competitive antagonistic effect was demonstrated in nicotinic acetylcholine receptors (a1bεd, Torpedo, a1bgd, a3b4a55b2, a3b2, a4b2, a7) of the Cys-loop superfamily providing an alternate pharmacological pathway. Our laboratory has shown that another cationselective member of the Cys-loop superfamily, the serotonin type 3 receptor (5-HT 3 -R), is modulated by bupropion at clinically relevant concentrations. Specifically, we determined that bupropion acts as a non-competitive antagonist at 5-HT 3A subunits. 5-HT 3 -Rs are found pre-and postsynaptically, and are currently targeted by anti-emetics and irritable bowel syndrome treatments. They also hold promise as potential future targets for multiple neurological disorders, such as Alzheimer's disease, schizophrenia, and bipolar disorder. The 5-HT 3 -R family consists of five different subunits (A-E) but the assembly of this receptor requires the 3A subunit, yielding either a homomer or heteromer with another subunit. To date, only the interaction of bupropion with the 3A subunit has been studied. Here, we extend our investigations to heteromeric 5-HT 3AB -Rs, which are found in the central and peripheral nervous system, predominantly in the amygdala, caudate nucleus, and hippocampus. The functional interaction of bupropion with 5-HT 3AB was characterized in Xenopus oocytes using two-electrode voltage clamp and patch clamp techniques. Docking studies and site-directed mutagenesis were used to identify the binding site/s in 5-HT 3 -R. Our studies confirmed that bupropion, similar to other non-competitive antagonists, evokes different responses in 5-HT 3AB -Rs as compared to the homomeric 5-HT 3A -Rs.